Methods for generating or increasing revenues from crops

ABSTRACT

The present invention provides methods of doing business and providing services. For example, methods of increasing the revenue of crops are provided. To this end, the method includes the use of a nucleic acid sequences of plant centromeres. This will permit construction of stably inherited recombinant DNA constructs and mini chromosomes which can serve as vectors for the construction of transgenic plant and animal cells.

This application is a continuation of U.S. patent application Ser. No.10/170,944, filed Jun. 12, 2002, which is a continuation-in-part of U.S.patent application Ser. No. 09/531,120, filed Mar. 17, 2000, each ofwhich disclosures is specifically incorporated herein by reference inits entirety.

The file copy of the sequence listing is submitted on a Compact-DiscRead Only Memory (CD-ROM). The sequence listing is saved as an ASCII DOStext file named 30001A.txt (4 KB), which was created on Feb. 2, 2007.The contents of the CD-ROM are incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

The present invention relates generally to methods of doing business.More specifically, the present invention relates to methods ofgenerating and/or increasing the revenue derived from crops.

To improve agricultural traits or produce certain products, it has beenknown to add a gene to an organism such as a plant. Although genes, fora vast array of products, have been proposed or identified, thetechnologies in the industry, heretofore, have severely curtaileddelivery of several genes into a plant.

Two general approaches are used for introduction of new geneticinformation (“transformation”) into cells. One approach is to introducethe new genetic information as part of another DNA molecule, referred toas an “episomal vector,” or “mini chromosome”, which can be maintainedas an independent unit (an episome) apart from the host chromosomal DNAmolecule(s). Episomal vectors contain all the necessary DNA sequenceelements required for DNA replication and maintenance of the vectorwithin the cell. Many episomal vectors are available for use inbacterial cells (for example, see Maniatis et al., 1982). However, onlya few episomal vectors that function in higher eukaryotic cells havebeen developed. Higher eukaryotic episomal vectors were primarily basedon naturally occurring viruses. In higher plant systems gemini virusesare double-stranded DNA viruses that replicate through a double-strandedintermediate upon which an episomal vector could be based, although thegemini virus is limited to an approximately 800 bp insert. Although anepisomal plant vector based on the Cauliflower Mosaic Virus has beendeveloped, its capacity to carry new genetic information also is limited(Brisson et al., 1984).

The other general method of genetic transformation involves integrationof introduced DNA sequences into the recipient cell's chromosomes,permitting the new information to be replicated and partitioned to thecellos progeny as a part of the natural chromosomes. The introduced DNAusually is broken and joined together in various combinations before itis integrated at random sites into the cell's chromosome (see, forexample Wigler et al., 1977). Common problems with this procedure arethe rearrangement of introduced DNA sequences and unpredictable levelsof expression due to the location of the transgene in the genome or socalled “position effect variation” (Shingo et al., 1986). Further,unlike episomal DNA, integrated DNA cannot normally be preciselyremoved. A more refined form of integrative transformation can beachieved by exploiting naturally occurring viruses that integrate intothe host's chromosomes as part of their life cycle, such as retroviruses(see Cepko et al., 1984). In mouse, homologous integration has recentlybecome common, although it is significantly more difficult to use inplants (Lam et al. 1996).

The most common genetic transformation method used in higher plants isbased on the transfer of bacterial DNA into plant chromosomes thatoccurs during infection by the phytopathogenic soil bacteriumAgrobacterium (see Nester et al., 1984). By substituting genes ofinterest for the naturally transferred bacterial sequences (calledT-DNA), investigators have been able to introduce new DNA into plantcells. However, even this more “refined” integrative transformationsystem is limited in three major ways. First, DNA sequences introducedinto plant cells using the Agrobacterium T-DNA system are frequentlyrearranged (see Jones et al., 1987). Second, the expression of theintroduced DNA sequences varies between individual transformants (seeJones et al., 1985). This variability is presumably caused by rearrangedsequences and the influence of surrounding sequences in the plantchromosome (i.e., position effects), as well as methylation of thetransgene. A third drawback of the Agrobacterium T-DNA system is thereliance on a “gene addition” mechanism: the new genetic information isadded to the genome (i.e., all the genetic information a cell possesses)but does not replace information already present in the genome.

One attractive alternative to commonly used methods of transformation isthe use of an artificial chromosome. Artificial chromosomes are man-madelinear or circular DNA molecules constructed from cis-acting DNAsequence elements that provide replication and partitioning of theconstructed chromosomes (see Murray et al., 1983). Desired elementsinclude: (1) Autonomous Replication Sequences (ARS) (these haveproperties of replication origins, which are the sites for initiation ofDNA replication), (2) Centromeres (site of kinetochore assembly andresponsible for proper distribution of replicated chromosomes at mitosisor meiosis), and (3) if the chromosome is linear, telomeres (specializedDNA structures at the ends of linear chromosomes that function tostabilize the ends and facilitate the complete replication of theextreme termini of the DNA molecule).

The essential chromosomal elements for construction of artificialchromosomes have been precisely characterized in lower eukaryoticspecies, and more recently in mouse and human. ARSs have been isolatedfrom unicellular fungi, including Saccharomyces cerevisiae (brewer'syeast) and Schizosaccharomyces pombe (see Stinchcomb et al., 1979 andHsiao et al., 1979). An ARS behaves like a replication origin allowingDNA molecules that contain the ARS to be replicated as an episome afterintroduction into the cell nuclei of these fungi. DNA moleculescontaining these sequences replicate, but in the absence of a centromerethey are partitioned randomly into daughter cells.

Artificial chromosomes have been constructed in yeast using the threecloned essential chromosomal elements. Murray et al., 1983, disclose acloning system based on the in vitro construction of linear DNAmolecules that can be transformed into yeast, where they are maintainedas artificial chromosomes. These yeast artificial chromosomes (YACs)contain cloned genes, origins of replication, centromeres and telomeresand are segregated in daughter cells with high fidelity when the YAC isat least 100 kB in length. Smaller CEN-containing vectors may be stablysegregated, however, when in circular form.

None of the essential components identified in unicellular organisms,however, function in higher eukaryotic systems. For example, a yeast CENsequence will not confer stable inheritance upon vectors transformedinto higher eukaryotes. While such DNA fragments can be readilyintroduced, they do not stably exist as episomes in the host cell. Thishas seriously hampered efforts to produce artificial chromosomes inhigher organisms.

In one case, a plant artificial chromosome was discussed (Richards etal., U.S. Pat. No. 5,270,201). However, this vector was based on planttelomeres, as a functional plant centromere was not disclosed. Whiletelomeres are important in maintaining the stability of chromosomaltermini, they do not encode the information needed to ensure stableinheritance of an artificial chromosome. It is well documented thatcentromere function is crucial for stable chromosomal inheritance inalmost all eukaryotic organisms (reviewed in Nicklas 1988). For example,broken chromosomes that lack a centromere (acentric chromosomes) arerapidly lost from cell lines, while fragments that have a centromere arefaithfully segregated. The centromere accomplishes this by attaching,via centromere binding proteins, to the spindle fibers during mitosisand meiosis, thus ensuring proper gene segregation during celldivisions.

In contrast to the detailed studies done in S. cerevisiae and S. pombe,less is known about the molecular structure of functional centromere DNAof higher eukaryotes. Ultrastructural studies indicate that highereukaryotic kinetochores, which are specialized complexes of proteinsthat form on the chromosome during late prophase, are large structures(mammalian kinetochore plates are approximately 0.3 μm in diameter)which possess multiple microtubule attachment sites (reviewed in Rieder,1982). It is therefore possible that the centromeric DNA regions ofthese organisms will be correspondingly large, although the minimalamount of DNA necessary for centromere function may be much smaller.

The above studies have been useful in elucidating the structure andfunction of centromeres. The extensive literature indicating both thenecessity of centromeres for stable inheritance of chromosomes, and thenon-functionality of yeast centromeres in higher organisms, demonstratethat cloning of a functional centromere from a higher eukaryote is anecessary first step in the production of artificial chromosomessuitable for use in higher plants and animals. The production ofartificial chromosomes with centromeres which function in highereukaryotes would overcome many of the problems associated with the priorart and represent a significant breakthrough in biotechnology research.

SUMMARY OF THE INVENTION

The present invention provides methods for improving crops as well asreducing the time necessary to produce new crops. Pursuant to thepresent invention, methods are provided that allow one to increaserevenues associated with crops, develop new crops, develop new avenuesfor generating revenues from crops, and provide new services to a thirdparty.

By allowing for the isolation and identification of plant centromere DNAsequences from the total genomic DNA of an organism or fractions thereofit is possible to construct chromosomes having functional centromeresand carrying large number of genes. As noted above, genes for producinga vast set of products have been identified, but technologies usedwithin the industry severely limit the delivery of these genes to plantcells. One or at most a few genes are typically inserted into randomlocations in the host chromosomes, which can irreversibly disrupt hostgene functions while causing variable and uncontrolled expression of theintroduced genes. The present invention makes it possible to overcomethe technical limitations associated with gene delivery in crop species,thereby allowing for the ability to shorten the time required for cropdevelopment.

To this end, in an embodiment, the present invention provides a methodfor providing a service. The method comprises the steps of: identifyinga sequence associated with a centromere of a crop; using a gene or anumber of genes and the sequence to create a minichromosome; andintroducing the minichromosome into a cell of the commercial plant tocreate a transgenic plant having a desirable characteristic not presentin the crop.

In an embodiment, the sequence is isolated after it is identified.

In an embodiment, the commercial plant is chosen from the groupconsisting of vegetable crop, fruit and vine crops, and field cropplants.

In an embodiment, the desirable characteristic is chosen from the groupconsisting of insect resistances, disease resistances, herbicideresistances, environmental resistances, agronomic characteristics,nutrient utilization, nutrient content, production of bioproducts,production of pharmaceutical products, production of chemical products,chemical assimilation, and reduction of length to maturity.

In an embodiment, the gene is selected from the group consisting of aselectable marker gene, a screenable marker gene, an antibioticresistance gene, a ligand gene, an enzyme gene, a herbicide resistancegene, a nitrogen fixation gene, a plant pathogen defense gene, a plantstress-induced gene, a toxin gene, a receptor gene, an interleukin gene,a clotting factor gene, a cytokine gene, a growth factor gene, a geneencoding an enzyme, a gene encoding an antibody, a gene encoding anantigen for a vaccine, a transcription factor, a cytoskeletal protein, aDNA-binding protein, a protease, an endonuclease, a lipid, a seedstorage gene, and a biosynthetic gene for producing pharmaceuticallyactive proteins, small molecules with medicinal properties, chemicalswith industrial utility, nutraceuticals, carbohydrates, RNAs, lipids,fuels, dyes, pigments, vitamins, scents, flavors, vaccines, antibodies,and hormones.

In an embodiment, the new commercial plant has at least two desirablecharacteristics not present in the commercial plant.

In an embodiment, the minichromosome includes at least two genes eachproducing a different desirable characteristic.

In an embodiment, the method includes the step of producing seeds thatgenerate the new commercial plant.

In an embodiment, the method includes the step of extracting the desiredproduct from the new commercial plant.

In a further embodiment, the present invention provides a method ofincreasing revenues from a species of plant. The method comprises thesteps of: identifying a sequence associated with a centromere of thespecies of plant; using a gene or a number of genes and the sequence tocreate a minichromosome; and introducing the minichromosome into thespecies of plant to create a plant having a desirable characteristic notnaturally present in the species plant or to increase or decrease thelevel of a naturally occurring characteristic.

In yet another embodiment of the present invention, a method forincreasing the value of a specific crop is provided. The methodcomprises the steps of: identifying a sequence associated with acentromere of the crop; using a gene or a number of genes and thesequence to create a minichromosome; and introducing the minichromosomeinto the crop to create a transgenic plant having a characteristic thatcauses the modified crop to be more valuable.

Still further, in an embodiment, a method for increasing crop yield isprovided. The method comprises the steps of: identifying a sequenceassociated with a centromere of the crop; using a gene or a number ofgenes and the sequence to create a minichromosome; and introducing theminichromosome into the crop to create a transgenic plant having adesirable characteristic not naturally present in the crop or toincrease or decrease the level of a naturally occurring characteristic.

Moreover, in an embodiment, the present invention provides a method forproducing a new seed. The method includes the steps of: identifying asequence associated with a centromere of the commercial plant; using agene or a number of genes and the sequence to create a minichromosome;introducing the minichromosome into the commercial plant to create atransgenic plant having a desirable characteristic not present in thecommercial plant or to increase or decrease the level of a naturallyoccurring characteristic; allowing the transgenic plant to mature; andcollecting seeds from the mature plant.

Further, the present invention provides a method for generating revenuebased on agricultural products. The method comprises the steps of:developing a new crop based on a minichromosome comprising a sequence ofa centromere; and receiving a fee for the new crop.

In an embodiment, the fee is received as a royalty payment.

In an embodiment, the fee is received through sales of seed generated bythe new crop.

In an embodiment, the fee is based on sales of a product made from thenew crop.

In an embodiment, the fee is based on licensing of the new crop orproduct.

In an embodiment, the fee is based on the receipt of products orservices.

In addition, in an embodiment, the present invention provides a methodfor performing a service comprising the steps of: developing a modifiedplant having a desired characteristic that does not naturally occur in anon-modified plant by inserting into a cell of the non-modified plant aminichromosome comprising a sequence of a centromere and a geneexpressing the desired characteristic.

In a still further embodiment, the present invention provides a methodfor performing a service for a third party including the step of:isolating from a total genomic DNA of a plant a nucleotide sequencerelating to the plant's centromere and using the nucleotide sequence tomake a minichromosome for the third party.

Yet another embodiment of the present invention provides methods forestablishing strategic relationships with a third party. The method cancomprise the steps of initiating a research and development program witha third party to develop a crop that includes a mini chromosomecomprising a gene and a centromere sequence.

If desired, the method can include the step of working with the thirdparty to develop the crop.

In an embodiment, the method includes the step of licensing to the thirdparty at least a portion of the technology necessary to develop thecrop.

In order to obtain a centromere DNA sequence from a selected organism,in an embodiment, the method that is used comprises the steps ofpreparing a sample of genomic DNA from a selected organism, obtaining aplurality of nucleic acid segments from the genomic DNA and screeningthe nucleic acid segments to identify one or more centromere nucleicacid sequences. In an embodiment, the method of obtaining the pluralityof nucleic acid segments comprises contacting said genomic DNA with arestriction endonuclease and selecting nucleic acid segments containingrepetitive DNA to obtain said plurality of nucleic acid segments. Inanother embodiment, the method of obtaining the plurality of nucleicacid segments comprises contacting said genomic DNA with a methylationsensitive restriction endonuclease and selecting nucleic acid segmentsexhibiting resistance to cleavage with said methylation sensitiverestriction endonuclease to obtain said plurality of nucleic acidsegments. In yet another embodiment, the method of obtaining theplurality of nucleic acid segments comprises contacting said genomic DNAwith a restriction endonuclease or physically shearing said genomic DNAand selecting nucleic acid segments that anneal rapidly afterdenaturation to obtain said plurality of nucleic acid segments.

In another aspect, the invention provides a method for identifying acentromere nucleic acid sequence from a dataset of the genomic sequencesof an organism. The method comprises the steps of (1) providing a firstdataset consisting of the genomic sequences, or a representativefraction of genomic sequence, of the organism; (2) identifying andeliminating known non-centromeric repeat sequences from the firstdataset by using the BLAST sequence-comparison algorithm to create asecond dataset; (3) comparing each sequence in the second dataset toitself by using the BLAST sequence comparison algorithm, obtaining aBLAST score for each pair of sequence compared, and collecting highscore pairs to create a third dataset; (4) examining the BLAST score ofeach high score pair in the third dataset and eliminating the pairshaving a score greater than 10⁻²⁰ to create a fourth dataset; (5)eliminating the high score pairs in the fourth dataset having less than80 bp or more than 250 bp to create a fifth dataset; (6) examining thenucleotide position of each high score pair in the fifth dataset andeliminating pairs having 100% identity as well as identical nucleotidepositions to create a sixth dataset; (7) examining the nucleotideposition of each high score pair in the sixth dataset and eliminatingpairs having opposite orientation of the nucleotides to create a seventhdataset; (8) examining the nucleotide position of both sequences foreach high score pair in the seventh dataset and eliminating sequencesthat are overlapping to create an eighth dataset; and (9) examining thenucleotide position of each sequence in the eighth dataset andeliminating sequences not having at least one neighboring sequencewithin 250 bp to create a ninth dataset; and (10) comparing eachsequence in the ninth dataset to all other sequences in the ninthdataset by using the BLAST sequence comparison algorithm and selectingthe most common sequence as a centromere sequence of the organism. Inone embodiment, the known non-centromeric repeat sequence in the secondstep is a ribosomal DNA.

A number of centromeres can be used pursuant to the present invention.In an aspect of the present invention, a Brassica oleracea centromerecomprising Brassica oleracea centromere DNA is used. In one embodiment,the Brassica oleracea centromere is defined as comprising n copies of arepeated nucleotide sequence, wherein n is at least 2. Potentially anynumber of repeat copies capable of physically being placed on therecombinant construct could be included on the construct, includingabout 5, 10, 15, 20, 30, 50, 75, 100, 150, 200, 300, 400, 500, 750,1,000, 1,500, 2,000, 3,000, 5,000, 7,500, 10,000, 20,000, 30,000,40,000, 50,000, 60,000, 70,000, 80,000, 90,000 and about 100,000,including all ranges in-between such copy numbers. In one embodiment,the repeated nucleotide sequence is isolated from Brassica oleraceagiven by SEQ ID NO:1, 2, 3, or 4.

In yet another aspect, a Glycine max centromere comprising glycine maxcentromere DNA is used. In an embodiment, the Glycine max centromere isdefined as comprising n copies of a repeated nucleotide sequence,wherein n is at least 2. Potentially any number of repeat copies capableof physically being placed on the recombinant construct could beincluded on the construct, including about 5, 10, 15, 20, 30, 50, 75,100; 150, 200, 300, 400, 500, 750, 1,000, 1,500, 2,000, 3,000, 5,000,7,500, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000,90,000 and about 100,000, including all ranges in-between such copynumbers. In one embodiment, the repeated nucleotide sequence is isolatedfrom Glycine max given by SEQ ID NO:5, 6, 7, or 8.

In yet another aspect, a Lycopersicon esculentum centromere comprisingLycopersicon esculentum centromere DNA is used. In an embodiment, theLycopersicon esculentum centromere is defined as comprising n copies ofa repeated nucleotide sequence, wherein n is at least 2. Potentially anynumber of repeat copies capable of physically being placed on therecombinant construct could be included on the construct, includingabout 5, 10, 15, 20, 30, 50, 75, 100, 150, 200, 300, 400, 500, 750,1,000, 1,500, 2,000, 3,000, 5,000, 7,500, 10,000, 20,000, 30,000,40,000, 50,000, 60,000, 70,000, 80,000, 90,000 and about 100,000,including all ranges in-between such copy numbers. In one embodiment,the repeated nucleotide sequence is isolated from Lycopersiconesculentum given by SEQ ID NO:9 or 10.

In yet another aspect, a Zea mays centromere comprising Zea mayscentromere DNA is used. In an embodiment, the centromere is defined ascomprising n copies of a repeated nucleotide sequence, wherein n is atleast 2. Potentially any number of repeat copies capable of physicallybeing placed on the recombinant construct could be included on theconstruct, including about 5, 10, 15, 20, 30, 50, 75, 100, 150, 200,300, 400, 500, 750, 1,000, 1,500, 2,000, 3,000, 5,000, 7,500, 10,000,20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 and about100,000, including all ranges in-between such copy numbers. In oneembodiment, the repeated nucleotide sequence is isolated from Zea maysgiven by SEQ ID NO:11, 12 or 13.

In yet another aspect, recombinant DNA construct comprising a plantcentromere sequence is used. The recombinant DNA construct mayadditionally comprise any other desired sequences, for example, atelomere. Still further, one may wish to include a structural gene onthe construct, or multiple genes. Examples of structural genes one maywish to use include a selectable or screenable marker gene, anantibiotic resistance gene, a ligand gene, an enzyme gene, a herbicideresistance gene, a nitrogen fixation gene, a plant pathogen defensegene, a plant stress-induced gene, a toxin gene, a receptor gene, a geneencoding an enzyme, a gene encoding an antibody, a gene encoding anantigen for a vaccine, a transcription factor, a cytoskeletal protein, aDNA-binding protein, a protease, an endonuclease, a lipid, a seedstorage gene, an interleukin gene, a clotting factor gene, a cytokinegene, a growth factor gene and a biosynthetic gene for producingpharmaceutically active proteins, small molecules with medicinalproperties, chemicals with industrial utility, nutraceuticals,carbohydrates, RNAs, lipids, fuels, dyes, pigments, vitamins, scents,flavors, vaccines, antibodies, and hormones.

In still yet another aspect, a recombinant DNA construct comprising aplant centromere sequence and which is capable of being maintained as achromosome, wherein the chromosome is transmitted in dividing cells isused. The plant centromere may be from any plant or may be from anyother source of DNA or may be partially or entirely synthetic in origin.

In yet another aspect, a recombinant DNA construct comprising a plantcentromere sequence and which is a plasmid is used. The plasmid maycontain any desired sequences, such as an origin of replication. Theplasmid may also comprise a selection marker.

In still yet another aspect, a mini chromosome comprising a plantcentromere sequence and may also contain a telomere sequence is used.Any additional desired sequences may be added to the mini chromosome,such as an autonomous replicating sequence and a structural gene such asthose described above. The mini chromosome may comprise any of thecentromere compositions disclosed herein.

The mini chromosome also may contain “negative” selectable markers whichconfer susceptibility to an antibiotic, herbicide or other agent,thereby allowing for selection against plants, plant cells or cells ofany other organism of interest containing a mini chromosome. The minichromosome also may include genes or other sequences which control thecopy number of the mini chromosome within a cell. One or more structuralgenes also may be included in the mini chromosome. Specificallycontemplated as being useful will be as many structural genes as may beinserted into the mini chromosome.

In still yet another aspect, a cell transformed with a recombinant DNAconstruct comprising a plant centromere sequence is used. The cell maybe of any type, including a prokaryotic cell or eukaryotic cell. Wherethe cell is a eukaryotic cell, the cell may be, for example, a yeastcell or a higher eukaryotic cell, such as plant cell. The plant cell maybe from a dicotyledonous plant, such as tobacco, tomato, potato,soybean, canola, sunflower, alfalfa, cotton and Arabidopsis, or may be amonocotyledonous plant cell, such as wheat, maize, rye, rice, turfgrass,oat, barley, sorghum, millet, and sugarcane. In one embodiment of theinvention, the plant centromere is a centromere chosen from the groupconsisting of Brassica oleracea, Glycine max, Lycopersicon esculentum,and Zea mays and the cell may be a cell chosen from one of the abovespecies or any other species. The recombinant DNA construct may compriseadditional sequences, such as a telomere, an autonomous replicatingsequence (ARS), a structural gene or genes, or a selectable orscreenable marker gene or genes, including as many of such sequences asmay physically be placed on said recombinant DNA construct. In oneembodiment of the invention, the cell is further defined as capable ofexpressing said structural gene. In another embodiment of the invention,a plant is provided comprising the aforementioned cells.

In still yet another aspect, a method for preparing a transgenic plantcell is used. The method comprises the steps of contacting a startingplant cell with a recombinant DNA construct comprising a plantcentromere sequence of the present invention, whereby the starting plantcell is transformed with the recombinant DNA construct.

In still yet another aspect, a transgenic crop comprising a minichromosome, wherein the mini chromosome comprises a plant centromeresequence of the present invention is used. The mini chromosome mayfurther comprise a telomere sequence, an autonomous replicating sequenceor a structural gene, such as a selectable or screenable marker gene, anantibiotic resistance gene, a ligand gene, an enzyme gene, a herbicideresistance gene, a nitrogen fixation gene, a plant pathogen defensegene, a plant stress-induced gene, a toxin gene, a receptor gene, a geneencoding an enzyme, a gene encoding an antibody, a gene encoding anantigen for a vaccine, a transcription factor, a cytoskeletal protein, aDNA-binding protein, a protease, an endonuclease, a lipid, a seedstorage gene, an interleukin gene, a clotting factor gene, a cytokinegene, a growth factor gene and a biosynthetic gene for producingpharmaceutically active proteins, small molecules with medicinalproperties, chemicals with industrial utility, nutraceuticals,carbohydrates, RNAs, lipids, fuels, dyes, pigments, vitamins, scents,flavors, vaccines, antibodies, and hormones. The transgenic crop may beany type of crop, such as a dicotyledonous plant, for example, tobacco,tomato, potato, pea, carrot, cauliflower, broccoli, soybean, canola,sunflower, alfalfa, cotton and Arabidopsis, or may be a monocotyledonousplant, such as wheat, maize, rye, rice, turfgrass, oat, barley, sorghum,millet, and sugarcane.

In still yet another aspect, the method includes preparing a transgeniccrop tissue. The method comprises the steps of contacting a startingcrop tissue with a recombinant DNA construct comprising a plantcentromere sequence of the present invention, whereby the starting croptissue is transformed with the recombinant DNA construct.

In still yet another aspect, the method includes preparing a transgeniccrop seed. The method comprises the steps of contacting a starting crop,crop tissue, or crop cell, with a recombinant DNA construct comprising aplant centromere sequence of the present invention, whereby the startingcrop, crop tissue, or crop cell is transformed with the recombinant DNAconstruct. These transformed crops, crop tissues, or crop cells areallowed to develop into mature crops, using standard agriculturaltechniques. Transgenic seed is then collected from these crops.

In still yet another aspect, the method includes preparing an extract ofa transgenic crop, crop tissue, crop seed, or crop cell. The methodcomprises the steps of contacting a starting crop, crop tissue, or cropcell with a recombinant DNA construct comprising a plant centromeresequence of the present invention, whereby the starting crop cell istransformed with the recombinant DNA construct. The resulting transgeniccrop, crop tissue, crop seed, or crop cell is then extracted andprocessed to yield the desirable product. One preferred desirableproduct is a food product. Another preferred desirable product is apharmaceutical product. Yet another preferred desirable product is achemical product.

Additional features and advantages of the present invention aredescribed in, and will be apparent from, the following DetailedDescription of the Invention and the figures.

BRIEF DESCRIPTION OF THE FIGURES

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1A-F Consensus sequences of repeats from Brassica oleracea. FIG. 1Ais the consensus sequence of ChrBo1 (SEQ ID NO:1). This consensus wasassembled from 33 sequences collected by the inventors. The length ofthis repeat is 180±0.86 base pairs, and A and T compose 60% of theconsensus. FIG. 1B is the consensus sequence of ChrBo2. This consensuswas assembled from 7 sequences, collected by the inventors. The lengthof this repeat is 180±0.45 base pairs, and A and T compose 63% of theconsensus. FIG. 1C is a comparison of the consensus sequences of ChrBo1(SEQ ID NO:1) and ChrBo2 (SEQ ID NO:2). The two repeats (ChrBo1 andChrBo2) were aligned to each other using the ClustalX program (ClustalXis a free multiple sequence alignment program for Windows). Those siteswith significant differences between the two sequences (Chi-squared,P<0.05) are highlighted. FIG. 1D is a revised consensus sequence ofChrBo1 (SEQ ID NO:3). This consensus was assembled from 33 DNA sequencescollected by the inventors and 18 sequences from Genbank, identified bythe accession numbers:

M30962 M30963 M31436 M31435 M31438 M31434 M31439 M31437 X68786 X12736X07519 X16589 X15291 X68783 X68784 X61583 AJ228348 Z22947

FIG. 1E is a revised consensus sequence of ChrBo2 (SEQ ID NO:6). Thisconsensus was assembled from 7 DNA sequences collected by the inventorsand 5 sequences from Genbank, identified by the accession numbersAJ228347, M30962, X12736, X61583, and X68785. FIG. 1F is a comparison ofthe revised consensus sequences of ChrBo1 and ChrBo2, aligned as forFIG. 1C.

FIG. 2A-F Consensus sequences of repeats from Glycine max. FIG. 2 A is aconsensus sequence of ChrGm1 (SEQ ID NO:5). This consensus was assembledfrom 32 sequences collected by the inventors. The length of this repeatis 92±0.79 base pairs, and A and T compose 63% of the consensus. FIG. 2Bis a consensus sequence of ChrGm2 (SEQ ID NO:6). This consensus wasassembled from 21 sequences collected by the inventors. The length ofthis repeat is 91±048 base pairs, and A and T compose 62% of theconsensus. FIG. 2C is a comparison of the consensus sequences of ChrGm1and ChrGm2. The two repeats (ChrGm1 and ChrGm2) were aligned to eachother using the ClustalX program. Those sites with significantdifferences between the two sequences (Chi-squared, P<0.05) arehighlighted.

FIG. 2D is a revised consensus sequence of ChrGm1 (SEQ ID NO:7). Thisconsensus was assembled from 32 DNA sequences collected by the inventorsand 1 sequence from Genbank, identified by the accession number Z26334.FIG. 2E is a revised consensus sequence of ChrGm2 (SEQ ID NO:8). Thisconsensus was assembled from 21 DNA sequences collected by the inventorsand 13 sequences from Genbank, identified by the accession numbersAF297983, AF297984, and AF297985. FIG. 2F is a comparison of the revisedconsensus sequences of ChrGm1 and ChrGm2, aligned as for FIG. 2C.

FIG. 3A-B Consensus sequences of repeats from Lycopersicon esculentum.FIG. 3A is a consensus sequence of ChrLe1 (SEQ ID NO:9). This consensuswas assembled from 42 sequences collected by the inventors. The lengthof this repeat is 181±0.61 base pairs, and A and T compose 50% of theconsensus. FIG. 3B is a revised consensus sequence of ChrLe1 (SEQ IDNO:10). This consensus was assembled from 32 sequences collected by theinventors and 2 Genbank sequences identified by the accession numbersX87233 and AY007367.

FIG. 4A-C Consensus sequences of repeats from Zea mays. FIG. 4A is aconsensus sequence of ChrZm1 (SEQ ID NO:11). This consensus wasassembled from 38 sequences collected by the inventors. The length ofthis repeat is 180±1.15 base pairs, and A and T compose 56% of theconsensus. FIG. 4B is a revised consensus sequence of ChrZm1 (SEQ IDNO:12). This consensus was assembled from 38 sequences collected by theinventors and 26 sequences from Genbank, identified by the accessionnumbers:

M32521 M32522 M32523 M32524 M32525 M32526 M32527 M32528 M32529 M32530M32531 M32532 M32533 M32534 M32535 M32536 M32537 M32538 M35408 AF030934AF030935 AF030936 AF030937 AF030938 AF030939 AF030940

FIG. 4C is a consensus sequence of ChrZm2 (SEQ ID NO:13). This consensuswas assembled from 6 sequences collected from Genbank identified by theaccession numbers:

AF0789 AF078919 AF078920 AF0789 AF078922 AF078923

The length of this repeat is 158±1.6 base pairs and A and T compose 53%of the consensus.

FIG. 5 Mini chromosome containing centromere sequences as well as minichromosome vector sequences

FIG. 6 Mini chromosome construct formed by mini chromosome vectortailing method.

FIG. 7A-7N. Exemplary Mini chromosome vectors: The vectors shown in FIG.7A, FIG. 7B, FIG. 7E, FIG. 7F, FIG. 7I and FIG. 7J have an E. coliorigin of replication which can be high copy number, low copy number orsingle copy. In FIGS. 7A-7N, the vectors include a multiple cloning sitewhich can contain recognition sequences for conventional restrictionendonucleases with 4-8 bp specificity as well as recognition sequencesfor very rare cutting enzymes such as, for example, I-Ppo I, I-Cue I,PI-Tli, PI-Psp I, Not I, and PI Sce I. In FIG. 7A-7N, the centromere isflanked by Lox sites which can act as targets for the site specificrecombinase Cre. FIG. 7A. Shows an E. coli plant circular shuttle vectorwith a plant ARS. FIG. 7B. Shows a plant circular vector without a plantARS. The vector relies on a plant origin of replication function foundin other DNA sequences such as selectable or screenable markers. FIG.7C. Shows a yeast-plant circular shuttle vector with a plant ARS. Theyeast ARS is included twice, once on either side of multiple cloningsite to ensure that large inserts are stable. FIG. 7D. Shows ayeast-plant circular shuttle vector without a plant ARS. The vectorrelies on a plant origin of replication function found in other plantDNA sequences such as selectable markers. The yeast ARS is includedtwice, once on either side of the multiple cloning site to ensure thatlarge inserts are stable. FIG. 7E. Shows an E. coli-Agrobacterium-plantcircular shuttle vector with a plant ARS. Vir functions for T-DNAtransfer would be provided in trans by a using the appropriateAgrobacterium strain. FIG. 7F. Shows an E. coli-Agrobacterium-plantcircular shuttle vector without a plant ARS. The vector relies on aplant origin of replication function found in other plant DNA sequencessuch as selectable markers. Vir functions for T-DNA transfer would beprovided in trans by a using the appropriate Agrobacterium strain. FIG.7G. Shows a linear plant vector with a plant ARS. The linear vectorcould be assembled in vitro and then transferred into the plant by, forexample, mechanical means such as micro projectile bombardment,electroporation, or PEG-mediated transformation. FIG. 7H. Shows a linearplant vector without a plant ARS. The linear vector could be assembledin vitro and then transferred into the plant by, for example, mechanicalmeans such as micro projectile bombardment, electroporation, orPEG-mediated transformation. FIGS. 7I-7N. The figures are identical toFIGS. 7A-7F, respectively, with the exception that they do not containplant telomeres. These vectors will remain circular once delivered intothe plant cell and therefore do not require telomeres to stabilize theirends.

FIG. 8. Sequence features at Arabidopsis CEN2 (A) and CEN4 (B). Centralbars depict annotated genomic sequence of indicated BAC clones; black,genetically-defined centromeres; white, regions flanking thecentromeres. Sequences corresponding to genes and repetitive features,filled boxes (above and below the bars, respectively), are defined as inFIG. 11A-T; predicted nonmobile genes, red; genes carried by mobileelements, black; nonmobile pseudogenes, pink; pseudogenes carried bymobile elements, gray; retroelements, yellow; transposons, green;previously defined centromeric repeats, dark blue; 180 bp repeats, paleblue. Chromosome-specific centromere features include a largemitochondrial DNA insertion (orange; CEN2), and a novel array of tandemrepeats (purple; CEN4). Gaps in the physical maps (//), unannotatedregions (hatched boxes), and expressed genes (filled circles) are shown.

FIG. 9. Method for converting a BAC clone (or any other bacterial clone)into a mini chromosome. A portion of the conversion vector willintegrate into the BAC clone (or other bacterial clone of interest)either through non-homologous recombination (transposable elementmediated) or by the action of a site specific recombinase system, suchas Cre-Lox or FLP-FRT.

FIG. 10A-G. Method for converting a BAC clone (or any other bacterialclone) into a mini chromosome. The necessary selectable markers andorigins of replication for propagation of genetic material in E. coli,Agrobacterium and Arabidopsis as well as the necessary genetic loci forAgrobacterium mediated transformation into Arabidopsis are cloned into aconversion vector. Using Cre/loxP recombination, the conversion vectorsare recombined into BACs containing centromere fragments to form minichromosomes.

FIG. 11A-T. Properties of centromeric regions on chromosomes II and IVof Arabidopsis. (Top) Drawing of genetically-defined centromeres (grayshading, CEN2, left; CEN4, right), adjacent pericentromeric DNA, and adistal segment of each chromosome, scaled in Mb as determined by DNAsequencing (gaps in the grey shading correspond to gaps in the physicalmaps). Positions in cM on the Rl map (maintained at the NottinghamArabidopsis Stock Centre) and physical distances in Mb, beginning at thenorthern telomere and at the centromeric gap, are shown. (Bottom) Thedensity of each feature (FIGS. 11A-11T) is plotted relative to theposition on the chromosome in Mb. (FIG. 11A, 11K) cM positions formarkers on the Rl map (solid squares) and a curve representing thegenomic average of 1 cM/221 kb (dashed line). A single crossover 20within CEN4 in the Rl mapping population ( Somerville and Somerville,1999) may reflect a difference between male meiotic recombinationmonitored here and recombination in female meiosis. (FIGS. 11 B-11 E andFIGS. 11L-11O) The % of DNA occupied by repetitive elements wascalculated for a 100 kb window with a sliding interval of 10 kb. (FIGS.11B, 11L) 180 bp repeats; (FIGS. 11C, 11M) sequences with similarity toretroelements, including del, Tal, Tal1, copia, Athila, LINE, Ty3, TSCL,106B (Athila-like), Tat1, LTRs and Cinful; (FIGS. 11D, 11N) sequenceswith similarity to transposons, including Tag1, En/Spm, Ac/Ds, Tam1MuDR, Limpet, MITES and Mariner; (FIGS. 11E, 11O) previously describedcentromeric repeats including 163A, 164A, 164B, 278A, 11B7RE, mil67,pAT27, 160-, 180- and 500-bp repeats, and telomeric sequences. (FIGS.11F, 11P) % adenosine+thymidine was calculated for a 50 kb window with asliding interval of 25 kb (FIGS. 11G-11J, 11Q-11T). The number ofpredicted genes or pseudogenes was plotted over a window of 100 kb witha sliding interval of 10 kb. (FIGS. 11G, 11I, 11Q, US) predicted genes(FIGS. 11G, 11Q) and pseudogenes (FIGS. 11I, 11S) typically not found onmobile DNA 5 elements; (FIGS. 11H, 11J, 11R, 11T) predicted genes (FIGS.11H, 11R) and pseudogenes (FIGS. 11J, 11T) often carried on mobile DNA,including reverse transcriptase, transposase, and retroviralpolyproteins. Dashed lines indicate regions in which sequencing orannotation is in progress, annotation was obtained from GenBank records,from the Arabidopsis Genome Annotation Database (AGAD) that wasdeveloped by The Institute for Genomic Research (TIGR) database, and byBLAST comparisons to the database of repetitive Arabidopsis sequences(AtRepBase); though updates to annotation records may change individualentries, the overall structure of the region will not be significantlyaltered.

FIG. 12. Methods for converting a BAC clone containing centromere DNAinto a mini chromosome for introduction into plant cells. The specificelements described are provided for exemplary purposes and are notlimiting. A) diagram of the BAC clone, noting the position of thecentromere DNA, a site-specific recombination site (for example, lox P),and the F origin of replication. B) Conversion vector containingselectable and color markers (for example, 35S-Bar, nptII, LAT52-GUS,Scarecrow-GFP), telomeres, a site-specific recombination site (forexample, lox P), antibiotic resistance markers (for example, amp orspc/str), Agrobacterium T-DNA borders (Agro Left and Right) and originof replication (RiA4). The product of site specific recombination withthe Cre recombinase at the lox P sites yields a circular product withcentromeric DNA and markers flanked by telomeres. After mini-chromosometransformation into plants; subsequently, the left and right borderswill likely be removed by the plant cell and additional telomericsequence added by the plant telomerase.

FIG. 13A-B. Conservation of Arabidopsis centromere DNA. BAC clones(bars) used to sequence CEN2 (FIG. 13A) and CEN4 (FIG. 13B) areindicated; arrows denote the boundaries of the genetically-definedcentromeres. PCR primer pairs yielding products from only Columbia(filled circles) or from both Landsberg and Columbia (open circles);BACs encoding DNA with homology to the mitochondrial genome (gray bars);180 bp repeats (gray boxes); unsequenced DNA (dashed lines); and gaps inthe physical map (double slashes) are shown.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides numerous methods for doing business orproviding a service. These methods include increasing revenues generatedby crops, developing new crops, and improving existing crops. As notedabove, although it has been known to add a gene to improve theagricultural traits of a plant, this technology has been severelylimited. One, or at most a few genes, are typically inserted into randomlocations in the host chromosome. This technology thereby canirreversibly disrupt host gene functions, can cause variable anduncontrolled expression of the introduced genes, and is increasinglytime consuming and expensive as the number of genes introducedincreases.

The inventors have identified the centromeres of a number of cropsallowing them to overcome technical limitations associated with genedelivery in crops. This technology allows the inventors to developmethods for improving crops and developing new crops. Through thesemethods, the inventors are able to increase revenues associated with acrop, generate additional revenues from crops, provide a multitude ofservices associated with crops as well as produce new products fromcrops. This technology also affords the ability to perform a number ofservices for third parties (entities that request a service from anotherparty) based on crops.

For example, in an embodiment, the present invention allows theinventors to provide a service to a third party. The service includesidentifying a sequence associated with the centromere of a commercialplant. A gene or set of genes to be added to the commercial plant isthen chosen. The gene or set of genes and the sequence is then utilizedto create a mini chromosome. This mini chromosome is then introducedinto a cell of the commercial plant to create a transgenic plant havingdesirable characteristics not present in the commercial plant. A varietyof different genes can be utilized.

This method can be used to increase the revenues generated by commercialplants. In this regard, the desirable characteristic can be chosen sothat the resultant plant is more valuable. As used herein, the term“more valuable” can have a variety of meanings. In its most basic form,it means more revenue is generated by the crop. However, the term canalso mean that the costs associated with the crop are reduced. In thisregard, the gene or set of genes can add characteristics that make thecommercial plant more hearty (including but not limited tocharacteristics such as standility, draught tolerance, improved stature,cold tolerance, and pest resistance). On the other hand, the gene or setof genes can cause the plant to mature quicker. A variety ofcharacteristics can be added that can increase the value of thecommercial plant.

In an embodiment, a method of generating revenues based on agriculturalproducts is provided. The method includes the step of developing a newcrop based on a mini chromosome including a sequence of a centromere andreceiving a fee for the new crop. The term “crop” is defined herein inthe definitions section. As used herein, the term “new” as modifyingcrop means a crop, that includes a gene or set of genes or expresses atrait, that is in nature not typically part of the genetic sequence ofor expressed by the crop. Additionally, the term “new” as modifying cropcan also mean a crop that includes a gene or set of genes naturallyfound in the crop but modified so that their copy number is changed.Additionally, the term “new” as modifying crop can also mean a crop thatis modified to increase or decrease a trait that in nature is typicallyfound at undesirable levels. Thus, developing a “new soybean” means thata soybean is provided that includes a trait or a gene in its sequencethat is not typically found in soybeans in nature or a soybean that hasbeen modified to increase or decrease an existing trait or gene product.

A variety of mechanisms can be utilized as a method of receiving a feefor the new crop. Such methods can include a fee that is received as aroyalty payment. The royalty payment may take a variety of forms. Theroyalty can be charged either as a one-time payment, fixed fee paid on aregular basis, or a portion of the sales, or any combination thereof.For example, the technology for creating a new crop or the new cropitself may be licensed to a third party. The third party may sell seedsto a farmer or other commercial entity for a set price and the fee isset as a portion of the sales price. For example, a fee of 1% of thesales of the seeds can be charged as the fee. If desired, the fee can bebased on sales of a product made from the new crop, e.g., foods,pharmaceuticals, or chemicals. If desired, the fee can comprise, inwhole or in part, a cross-license of technology from a third party. Avariety of mechanisms can be used to meet the step receiving a fee.Essentially, all that is required is that a valve is received.

The present invention affords a variety of different business methods.In part, these methods are due to the ability to introduce a number andvariety of different genes into a crop. These genes can be introducedinto the plants in a single step. Because the genes can be introducedindependent of the host genome, this reduces the damaging effects thatoccur in transformation technologies. This independence allows theadditional advantage that the chromosome can be eliminated or modifiedwhen desired. Further, because the DNA sequences surrounding the genesare defined, copy number and expression levels of the genes can becontrolled precisely. This technology allows one to shorten both thecost and the time required for crop development.

Pursuant to the present invention, strategic relationships can be forgedwith third parties, e.g., agricultural companies that can benefit fromthe use of the technology in crops. These relationships can be used toinitiate and drive a research and development program using cropcentromeres to introduce engineered chromosomes into crops. Suchprograms can be based on the ability to use existing genes, or genes tobe developed, to produce a unique variety of seeds. It should be notedthat these programs can be used to not only generate improved crops, butalso to produce pharmaceuticals, chemicals, and compounds based, atleast in part, on crops.

In addition to the methods set forth above, it is also envisioned thatmethods of generating revenue can include, without limitation: sellingcentromeres, possibly for specific fields of use or exclusive use;developing chromosomes per customer specifications; designing plantscarrying custom chromosomes to enhance crop values; and producing highvalue products synthesized by genes carried on engineered chromosomes.

The methods of the present invention are premised, in part, on the factthat the inventors have overcome the deficiencies in the prior art byproviding the nucleic acid sequences of plant centromeres. Thesignificance of this achievement relative to the prior art isexemplified by the general lack of detailed information in the artregarding the centromeres of multicellular organisms in general. Todate, the most extensive and reliable characterization of centromeresequences has come from studies of lower eukaryotes such as S.cerevisiae and S. pombe, where the ability to analyze centromerefunctions has provided a clear picture of the desired DNA sequences. TheS. cerevisiae centromere consists of three essential regions, CDEI,CDEII, and CDEIII, totaling only 125 bp, or approximately 0.006 to 0.06%of each yeast chromosome (Carbon et al., 1990; Bloom 1993). S. pombecentromeres are between 40 and 100 kB in length and consist ofrepetitive elements that comprise 1 to 3% of each chromosome (Baum etal., 1994). Subsequent studies, using tetrad analysis to follow thesegregation of artificial chromosomes, demonstrated that less than ⅕ ofthe naturally occurring S. pombe centromere is sufficient for centromerefunction (Baum et al., 1994).

In contrast, the centromeres of mammals and other higher eukaryotes areless understood. Although DNA fragments that hybridize to centromericregions in higher eukaryotes have been identified, in many cases, littleis known regarding the functionality of these sequences (see Tyler-Smithet al., 1993). Centromere repeats often correlate with centromerelocation, with probes to the repeats mapping both cytologically andgenetically to centromere regions. Many of these sequences aretandemly-repeated satellite elements and dispersed repeated sequences inarrays ranging from 300 kB to 5000 kB in length (Willard 1990). To date,only one of these repeats, a 171 bp element known as the alphoidsatellite, has been shown by in situ hybridization to be present at eachhuman centromere (Tyler-Smith et al., 1993). Whether repeats themselvesrepresent functional centromeres remains controversial, as other genomicDNA can be required to confer efficient inheritance upon a region of DNA(Willard, 1997). Alternatively, the positions of some higher eukaryoticcentromeres have been estimated by analyzing the segregation ofchromosome fragments. This approach is imprecise, however, because alimited set of fragments can be obtained, and because normal centromerefunction is influenced by surrounding chromosomal sequences (forexample, see Koornneef, 1983; FIG. 2).

A more precise method for mapping centromeres that can be used in intactchromosomes is tetrad analysis (Mortimer et al., 1981), which provides afunctional definition of a centromere in its native chromosomal context.Centromeres that have been mapped in this manner include those from theyeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, andKluyveromyces lactis (Carbon et al., 1990; Hegemann et al., 1993). Inmany of these systems, accurate mapping of the centromeres made itpossible to clone centromeric DNA, using a chromosome walking strategy(Clarke et al., 1980). Subsequently, artificial chromosome assays wereused to define more precisely the centromere sequences (Hegemann et al.,1993; Baum et al., 1994).

Attempts to develop a reliable centromeric assay in mammals have yieldedambiguous results. For example, Hadlaczky et al., (1991) identified a 14kB human fragment that can, at low frequency, result in de novocentromere formation in a mouse cell line. In situ hybridizationstudies, however, have shown that this fragment is absent from naturallyoccurring centromeres, calling into question the reliability of thisapproach for testing centromere function (Tyler-Smith et al., 1993).Similarly, transfection of alphoid satellites into cell lines results inthe formation of new chromosomes, yet some of these chromosomes alsorequired host sequences that could contribute centromere activity (Haafet al., 1992; Willard, 1997). Further, the novel chromosomes can havealphoid DNA spread throughout their length yet have only a singlecentromeric constriction, indicating that a block of alphoid DNA alonemay be insufficient for centromere function (Tyler-Smith et al., 1993).

Although plant centromeres can be visualized easily in condensedchromosomes, they have not been characterized as extensively ascentromeres from yeast or mammals. Genetic characterization has reliedon segregation analysis of chromosome fragments, and in particular onanalysis of trisomic strains that carry a genetically marked,telocentric fragment (for example, see Koornneef 1983). In addition,repetitive elements have been identified that are either genetically(Richards et al., 1991) or physically (Alfenito et al., 1993;Maluszynska et al., 1991) linked to a centromere. In no case, however,has the functional significance of these sequences been tested.

Cytology in Arabidopsis thaliana has served to correlate centromerestructure with repeat sequences. A fluorescent dye, DAPI, allowsvisualization of centromeric chromatin domains in metaphase chromosomes.A fluorescence in situ hybridization (FISH) probe based on 180 bp pAL1repeat sequences colocalized with the DAPI signature near thecentromeres of all five Arabidopsis chromosomes (Maluszynska et al.,1991; Martinez-Zapater et al., 1986). Although a functional role forpAL1 has been proposed, more recent studies have failed to detect thissequence near the centromeres in species closely related to Arabidopsisthaliana (Maluszynska et al., 1993). These results are particularlytroubling because one of the species tested, A. pumila, is thought to bean amphidiploid, derived from a cross between A. thaliana and anotherclose relative (Maluszynska et al., 1991; Price et al., 1995). Anotherrepetitive sequence, pAtT12, has been genetically mapped to within 5 cMof the centromere on chromosome 1 and to the central region ofchromosome 5 (Richards et al., 1991), although its presence on otherchromosomes has not been established. Like pAL1, a role for pAtT12 incentromere function remains to be demonstrated.

Due to the fact that kinetochores constitute a necessary link betweencentromeric DNA and the spindle apparatus, the proteins that areassociated with these structures recently have been the focus of intenseinvestigation (Bloom 1993; Earnshaw 1991). Human autoantibodies thatbind specifically in the vicinity of the centromere have facilitated thecloning of centromere-associated proteins (CENPs, Rattner 1991), and atleast one of these proteins belongs to the kinesin superfamily ofmicrotubule-based motors (Yen 1991). Yeast centromere-binding proteinsalso have been identified, both through genetic and biochemical studies(Bloom 1993; Lechner et al., 1991).

The centromeres of Arabidopsis thaliana have been mapped using trisomicstrains, where the segregation of chromosome fragments (Koornneef 1983)or whole chromosomes (Sears et al., 1970) was used to localize four ofthe centromeres to within 5, 12, 17 and 38 cM, respectively. Thesepositions have not been refined by more recent studies because themethod is limited the difficulty of obtaining viable trisomic strains(Koornneef 1983). These factors introduce significant error into thecalculated position of the centromere, and in Arabidopsis, where 1 cMcorresponds roughly to 200 kB (Koornneef 1987; Hwang et al., 1991), thismethod did not map any of the centromeres with sufficient precision tomake chromosome walking strategies practical. Mapping of the Arabidopsisgenome was also discussed by (Hauge et al., 1991).

I. Isolation of Centromere Clones

The present invention relates to methods of isolating and identifyingcentromere DNA sequences from total genomic DNA of an organism withoutgenetic mapping of the organism. Centromere DNA can be purified fromtotal genomic DNA using several methods which include: 1) digestinggenomic DNA with restriction enzymes and separating the fragments onagarose gels, to reveal major classes of repetitive DNA; 2) digestinggenomic DNA with restriction enzymes sensitive to DNA methylation andseparating the fragments on agarose gels to reveal the heavilymethylated fraction of the genome; and 3) collecting the rapidlyannealing fraction of denatured genomic DNA. These three methods isolatecentromere DNA; therefore, these methods are expected to independentlyisolate the same sequences, thus validating the sequences' centromereorigin. It is anticipated that each of these methods can be applied togenomic DNA from any organism, including some lower organisms such asyeasts, as well as higher organisms such as plants and animals. Each ofthese methods is described in detail below.

1. Isolation of repetitive DNA

Centromere regions often contain many copies of the same DNA sequence(repetitive DNA); such repeats can range in size from a few nucleotideslong to hundreds or thousands of bases. Such repetitive DNA can beidentified following digestion of genomic DNA with restrictionendonucleases. Digestion of non-repetitive genomic DNA with a particularrestriction enzyme produces a distribution of size fragments; incontrast, digestion of repeats with a restriction enzyme that cutswithin each repeat produces a fragment of a typical size. Thus, genomicDNA that has been cut with a restriction enzyme can be size fractionatedby agarose gel electrophoresis to reveal repetitive DNA elements; afterstaining the gel to reveal the DNA, the repetitive fragment can beexcised and purified using conventional techniques or commercial kits.Such repeats can be introduced into cloning vectors and characterized asdescribed below. By using this method with a variety of restrictionenzymes, different repetitive elements can be purified from genomic DNA.

2. Purification of Methylated DNA

This method is disclosed in detail in co-pending U.S. patent applicationSer. No. 09/888,220, filed Jun. 22, 2001, the disclosure of which isincorporated herein by reference in its entirety and made a part hereof.Plant centromere DNA is often extensively modified by methylation; thepresence of this methylation can be used to purify centromere fragments.Digestion of genomic DNA with a methylation-sensitive restrictionendonuclease (for example Sau3A or HpaII) yields a range of fragmentsizes; endonuclease sites that are methylated are protected fromdigestion. Heavily methylated DNA molecules, such as centromere DNA,yield large fragments after digestion and can therefore be separatedfrom the lightly or non-methylated fraction by virtue of their size. Forexample agarose gel electrophoresis, acrylamide gel electrophoresis,sucrose gradient fractionation, or other size fractionation techniquescan be used to separate these fragments into pools of “large” (7-12 kb)and “smaller” fragments (3-7 kb and 0-3 kb).

3. Isolation of Rapidly Annealing DNA.

The rapidity with which denatured single stranded DNA can reanneal withanother single stranded DNA molecule of complementary sequence uponrenaturation is dependant upon its abundance. Therefore when genomic DNAis denatured and allowed to renature, the repetitive fraction of thegenome, including centromere DNA, will renature before the unique andlow copy fractions of the genome. Thus by fragmenting purified genomicDNA, denaturing it, collecting fractions at specific time points (suchas 2, 4, 6, 8, and 10 minutes) during renaturation and treating thosefractions to remove unannealed DNA it is possible to purify repetitiveDNA from total genomic DNA. Several methods can be used to removeunannealed from annealed DNA including treatment of the sample with anenzyme, such as S1 nuclease, that degrades single-stranded DNA orexposure to an agent that binds single-stranded DNA such ashydroxylapatite. By varying the time at which fractions are collectedduring renaturation it is possible to separate DNA fragments into highlyrepetitive, moderately repetitive, and non-repetitive fractions.

II. Cloning and sequencing small fragments of centromere DNA

Repetitive or methylated DNA fragments isolated using the methodsdescribed above can be ligated (using T4 DNA ligase, for example) to aplasmid vector and cloned by transformation into E. coli. These clonescan then propagated, sequenced, used to assemble mini chromosomes, orused to identify larger centromere clones, generate molecular markersthat facilitate genetic mapping of centromeres, or create probes forchromosome mapping experiments such as fluorescent in situ hybridization(FISH).

III. Identifying centromere clones in genomic libraries

A genomic library can be screened for clones carrying centromere DNA byarraying the clones onto solid supports, such as membrane filters, andprobing with labeled fragments of purified centromere DNA, includingcloned repetitive or methylated DNA fragments described above, oralternatively, the entire set of rapidly annealing genomic DNA or highlymethylated genomic DNA fragments. Probes can be used singly or incombination. Typically these probes are labeled by incorporation ofradionucleotides, fluorescent nucleotides, or other chemical orenzymatic ligands that enable easy detection. The labeled probe DNA isdenatured and hybridized to the arrayed library using standard molecularbiology techniques. Hybridization is performed at a temperature thatwill discourage non-specific DNA annealing while promoting thehybridization of the labeled probe to complementary sequences. Afterincubation, the arrayed library is washed to remove unannealed probe,and a detection method appropriate to the label incorporated in theprobe is used. For example, if the probe is radiolabeled, the labeledfilter is exposed to X-ray film.

To identify centromere clones, the results of several hybridizationexperiments are quantitated and compared. In some cases, centromereclones may hybridize to only one probe; in other cases, the clones willhybridize to multiple probes. The hybridization intensity of each cloneto each probe can be measured and stored in a database. A preferredmethod for this analysis is to use software that digitizes thehybridization signals, assigns each signal to its corresponding cloneaddress, ensures that duplicate copies of the clones successfullyhybridized, and enters the resulting information into a relationaldatabase (MySQL for example). Another possible method for this analysisis to examine the hybridization results visually, estimate thehybridization intensity, and tabulate the resulting information.

The results of each hybridization experiment can be classified bygrouping clones that show hybridization to each probe above a thresholdvalue. For example, a computerized relational database can be queriedfor clones giving hybridization signals above a certain threshold forindividual probes or for multiple probes. Based on these hybridizationpatterns, clones can be grouped into categories, and representativemembers of each category can be tested in mini chromosomes.

IV. Identifying Centromere Sequences of an organism from genomicsequence Datasets

It is possible to devise computational algorithms to search databases ofgenomic sequences and select centromere sequences by identifying thosewith the characteristics of centromeres. For example, by selecting themost abundant tandem repeat of a particular size will yield centromeresequences. Other sets of characteristics could also be useful. Thefollowing is a computational algorithm designed to extract centromeresequences from genomic sequence datasets. It is important to note thatthis algorithm examines primary sequence data and does not rely on priorannotation of the sequence. The algorithm consists of steps 1 through10. However, not all the steps must occur in the listed order withoutaltering the output. Other rearrangements are easily recognizable by oneskilled in the art. The following terms are used in describing thealgorithm. BLAST is Basic Local Alignment Search Tool, a family offreely available algorithms for sequence database searches. BLAST alignstwo sequences and yields an estimate of the probability that thisalignment is significant, i.e. that it did not occur by chance. The twosequences compared by BLAST are called the ‘query’, usually a singlesequence of interest, and the ‘subject’, often part of a large databaseof sequences that are compared to the query. The query sequence (query)can also be part of a database of sequences. The outputs of BLAST areHigh Scoring Pairs (HSPs) that are alignments of subject and querysequences. Nucleotide position describes the position of a givennucleotide within the sequence, relative to the first nucleotide of thesequence. BLAST score (e value) is the likelihood that a given sequencealignment is significant (the lower the value the higher thesignificance). The algorithm is as follows:

(1) provide a first dataset consisting of the genomic sequences, or arepresentative fraction of genomic sequence, of the organism ofinterest;

(2) identify and eliminate known non-centromeric repeat sequences fromthe first dataset by using the BLAST sequence comparison algorithm tocreate a second dataset;

(3) compare each sequence in the second dataset to itself by using theBLAST sequence comparison algorithm, obtain a BLAST score for each pairof sequence compared, and collect high score pairs to create a thirddataset;

(4) examine the BLAST score of each high score pair in the third datasetand eliminate the pairs having a score greater than 10⁻²⁰ to create afourth dataset;

(5) eliminate the high score pairs in the fourth dataset having lessthan 80 bp or more than 250 bp to create a fifth dataset;

(6) examine the nucleotide position of each high score pair in the fifthdataset and eliminate pairs having 100% identity and identicalnucleotide positions (i.e. self matches) to create a sixth dataset;

(7) examine the nucleotide position of each high score pair in the sixthdataset and eliminate pairs having opposite orientation of thenucleotides to create a seventh dataset;

(8) examine the nucleotide position of both sequences for each highscore pair in the seventh dataset and eliminate sequences that areoverlapping to create an eighth dataset; and

(9) examine the nucleotide position of each sequence in the eighthdataset and eliminate sequences not having at least one neighboringsequence within 250 bp to create a ninth dataset; and

(10) compare each sequence in the ninth dataset to all other sequencesin the ninth dataset by using the BLAST sequence comparison algorithmand select the most common sequence as a centromere sequence of theorganism.

Optimally, the dataset used in step (1) in the above algorithm would bethe whole genome dataset such as the Arabidopsis genome which wasderived by methodical sequencing of mapped clones or the rice genomedataset which was derived by shotgun sequencing. Alternatively, thealgorithm would also work well on representative genome datasets. By theterm “representative genome datasets”, it is meant that the genomicsequences in the dataset is a subset of the sequences of the wholegenome collected from the whole genome without bias, such as bias towardcoding sequences. These sequences would be representative of the genomeas a whole. For example, the use of a 0.5× or even a 0.1× library ofArabidposis with representative genome datasets would return a truepositive result. On the contrary, the use of a subset of genomicsequences of the whole genome which are not representative of the wholegenome and biased toward certain sequences, such as the coding sequence,would return false positive results.

V. Centromere Compositions

The present invention concerns nucleic acid segments, isolatable fromvarious plant cells, that are enriched relative to total genomic DNA, orisolated from other sources or chemically synthesized with a novelsequence, or other nucleic acids that are capable of conferringcentromere activity to a recombinant molecule when incorporated into thehost cell. As used herein, the term “nucleic acid segment” refers to anucleic acid molecule that has been purified from total genomic nucleicacids of a particular species. Therefore, a nucleic acid segmentconferring centromere function refers to a nucleic acid segment thatcontains centromere sequences yet is isolated away from, or purifiedfree from, total genomic nucleic acids. Included within the term“nucleic acid segment”, are nucleic acid segments and smaller fragmentsof such segments, and also recombinant vectors, including, for example,mini chromosomes, artificial chromosomes, BACs, YACs, plasmids, cosmids,phage, viruses, and the like.

Similarly, a nucleic acid segment comprising an isolated or purifiedcentromeric sequence refers to a nucleic acid segment includingcentromere sequences and, in certain aspects, regulatory sequences,isolated substantially away from other naturally occurring sequences, orother nucleic acid sequences. In this respect, the term “gene” is usedfor simplicity to refer to a protein, polypeptide or peptide-encodingunit. As will be understood by those in the art, this functional termincludes both genomic sequences, cDNA sequences and smaller engineeredgene segments that may express, or may be adapted to express, proteins,polypeptides or peptides.

“Isolated substantially away from other sequences” means that thesequences of interest, in this case centromere sequences, are includedwithin the genomic nucleic acid clones provided herein. Of course, thisrefers to the nucleic acid segment as originally isolated, and does notexclude all genes or coding regions.

In particular embodiments, the invention concerns isolated nucleic acidsegments and recombinant vectors incorporating nucleic acid sequencesthat encode a centromere functional sequence that includes a contiguoussequence from the centromeres of the current invention. Again, nucleicacid segments that exhibit centromere function activity will be mostpreferred.

In still yet another aspect, the invention provides a plant centromerewhich is further defined as an Arabidopsis thaliana centromere. In yetanother embodiment of the invention, the plant centromere comprises anArabidopsis thaliana chromosome 2 centromere. The chromosome 2centromere may comprise, for example, from about 100 to about 611,000,about 500 to about 611,000, about 1,000 to about 611,000, about 10,000to about 611,000, about 20,000 to about 611,000, about 40,000 to about611,000, about 80,000 to about 611,000, about 150,000 to about 611,000,or about 300,000 to about 611,000 contiguous nucleotides of a firstnucleic acid sequence flanking a first series of 180 bp repeats incentromere 2 of A. thaliana. The centromere may also be defined ascomprising from about 100 to about 50,959, about 500 to about 50,959,about 1,000 to about 50,959, about 5,000 to about 50,959, about 10,000to about 50,959, 20,000 to about 50,959, about 30,000 to about 50,959,or about 40,000 to about 50,959 contiguous nucleotides of a secondnucleic acid sequence flanking a second series of 180 bp repeats incentromere 2 of A. thaliana. The centromere may comprise sequences fromboth of the third and the fourth sequences, including the aforementionedfragments, or the entirety of these sequences. In particularembodiments, the inventors contemplate a 3′ fragment of the firstsequence can be fused to a 5′ fragment of the second sequence,optionally including one or more 180 bp repeat sequence disposedtherebetween.

In still yet another aspect, the invention provides an Arabidopsisthaliana chromosome 4 centromere. In certain embodiments of theinvention, the centromere may comprise from about 100 to about1,082,000, about 500 to about 1,082,000, about 1,000 to about 1,082,000,about 5,000 to about 1,082,000, about 10,000 to about 1,082,000, about50,000 to about 1,082,000, about 100,000 to about 1,082,000, about200,000 to about 1,082,000, about 400,000 to about 1,082,000, or about800,000 to about 1,082,000 contiguous nucleotides of a third nucleicacid sequence flanking a third series of repeated sequences, includingcomprising the nucleic acid sequence of the third sequence. Thecentromere may also be defined as comprising from about 100 to about163,317, about 500 to about 163,317, about 1,000 to about 163,317, about5,000 to about 163,317, about 10,000 to about 163,317, about 30,000 toabout 163,317, about 50,000 to about 163,317, about 80,000 to about163,317, or about 120,000 to about 163,317 contiguous nucleotides of thenucleic acid sequence of a fourth sequence flanking a fourth series ofrepeated sequences, and may be defined as comprising the nucleic acidsequence of the fourth sequence. The centromere may comprise sequencesfrom both the third and the fourth sequences, including theaforementioned fragments, or the entirety of the third and the fourthsequences. In particular embodiments, the inventors contemplate a 3′fragment of the third sequence can be fused to a 5′ fragment of thefourth sequence, optionally including one or more 180 bp repeat sequencedisposed therebetween.

In yet another embodiment, there is provided a Arabidopsis thalianachromosome 1, 3 or 5 centromere selected from the nucleic acid sequencegiven by one of the repeated sequences in these chromosomes, orfragments thereof. The length of the repeat used may vary, but willpreferably range from about 20 bp to about 250 bp, from about 50 bp toabout 225 bp, from about 75 bp to about 210 bp, from about 100 bp toabout 205 bp, from about 125 bp to about 200 bp, from about 150 bp toabout 195 bp, from about 160 bp to about 190 and from about 170 bp toabout 185 bp including about 180 bp. In one embodiment, the constructcomprises at least 100 base pairs, up to an including the full length,of one of the preceding sequences. In addition, the construct mayinclude 1 or more 180 base pair repeats.

In one embodiment, the centromere n copies of a repeated nucleotidesequence obtained by the method disclosed herein, wherein n is at least2. Potentially any number of repeat copies capable of physically beingplaced on the recombinant construct could be included on the construct,including about 5, 10, 15, 20, 30, 50, 75, 100, 150, 200, 300, 400, 500,750, 1,000, 1,500, 2,000, 3,000, 5,000, 7,500, 10,000, 20,000, 30,000,40,000, 50,000, 60,000, 70,000, 80,000, 90,000 and about 100,000,including all ranges in-between such copy numbers. Moreover, the copies,while largely identical, can vary from each other. Such repeat variationis commonly observed in naturally occurring centromeres.

In another embodiment, the centromere is a Brassica oleracea centromerecomprising Brassica oleracea centromere DNA. In one embodiment, theBrassica oleracea centromere is defined as comprising n copies of arepeated nucleotide sequence, wherein n is at least 2. Potentially anynumber of repeat copies capable of physically being placed on therecombinant construct could be included on the construct, includingabout 5, 10, 15, 20, 30, 50, 75, 100, 150, 200, 300, 400, 500, 750,1,000, 1,500, 2,000, 3,000, 5,000, 7,500, 10,000, 20,000, 30,000,40,000, 50,000, 60,000, 70,000, 80,000, 90,000 and about 100,000,including all ranges in-between such copy numbers. In one embodiment,the repeated nucleotide sequence is isolated from Brassica oleraceagiven by SEQ ID NO:1, 2, 3, or 4.

In yet another embodiment, the centromere is a Glycine max centromerecomprising glycine max centromere DNA. In an embodiment, the Glycine maxcentromere is defined as comprising n copies of a repeated nucleotidesequence, wherein n is at least 2. Potentially any number of repeatcopies capable of physically being placed on the recombinant constructcould be included on the construct, including about 5, 10, 15, 20, 30,50, 75, 100, 150, 200, 300, 400, 500, 750, 1,000, 1,500, 2,000, 3,000,5,000, 7,500, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000,80,000, 90,000 and about 100,000, including all ranges in-between suchcopy numbers. In one embodiment, the repeated nucleotide sequence isisolated from Glycine max given by SEQ ID NO:5, 6, 7, or 8.

In yet another embodiment, the centromere is a Lycopersicon esculentumcentromere comprising Lycopersicon esculentum centromere DNA. In anembodiment, the Lycopersicon esculentum centromere is defined ascomprising n copies of a repeated nucleotide sequence, wherein n is atleast 2. Potentially any number of repeat copies capable of physicallybeing placed on the recombinant construct could be included on theconstruct, including about 5, 10, 15, 20, 30, 50, 75, 100, 150, 200,300, 400, 500, 750, 1,000, 1,500, 2,000, 3,000, 5,000, 7,500, 10,000,20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 and about100,000, including all ranges in-between such copy numbers. In oneembodiment, the repeated nucleotide sequence is isolated fromLycopersicon esculentum given by SEQ ID NO:9 or 10.

In yet another embodiment, the centromere is a Zea mays centromerecomprising Zea mays centromere DNA. In an embodiment, the centromere isdefined as comprising n copies of a repeated nucleotide sequence,wherein n is at least 2. Potentially any number of repeat copies capableof physically being placed on the recombinant construct could beincluded on the construct, including about 5, 10, 15, 20, 30, 50, 75,100, 150, 200, 300, 400, 500, 750, 1,000, 1,500, 2,000, 3,000, 5,000,7,500, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000,90,000 and about 100,000, including all ranges in-between such copynumbers. In one embodiment, the repeated nucleotide sequence is isolatedfrom Zea mays given by SEQ ID NO:11, 12 or 13.

The centromere can additionally be defined as the region of thechromosome where the sister chromatids pair during cell division. Thecentromere is also the chromosomal region where the kinetochore (thechromosomal attachment structure for the spindle) and the spindle (thecellular machinery that provides the motive force for chromosomesegregation) attach to the chromosome during mitosis and meiosis. Thecentromere is also defined as the region of the primary constriction ina condensed chromosome. The DNA of the centromere is characteristicallyheavily methylated, repetitive, and condensed (heterochromatic).

VI. Mini chromosome construction

Mini chromosomes are constructed by combining fragments of centromereDNA with other DNA sequences useful for propagation of the resultantrecombinant DNA molecule in E. coli, other bacteria, yeast or plants.Recombinant plasmids containing large fragments of centromere DNA arereferred to as centromere clones. Centromere sequences removed fromcentromere clones, or centromere sequences derived directly from genomicDNA, are referred to as centromere fragments. Recombinant constructscontaining DNA sequences necessary for the propagation, delivery,selection, and detection of mini chromosomes will be referred to as minichromosome vector sequences or mini chromosome vectors; these sequencescan include but are not limited to selectable marker genes, visiblemarker genes, origins of replication, restriction endonucleaserecognition sites, homing endonuclease recognition sites, sequencesrecognized by site specific recombinase enzymes, telomere sequences, andsequences required for delivery of mini chromosomes into bacteria, yeastor plant cells. Recombinant constructs containing both large centromerefragments as well as mini chromosome vector sequences are referred to asmini chromosomes. The process of assembling mini chromosomes fromcentromere clones/fragments and mini chromosome vector sequences can bedone in several ways, and involves techniques that are common practiceamong those trained in molecular biology:

1) Joining Centromere Fragments to Mini Chromosome Vector Sequences:

Centromere DNA fragments and mini chromosome vector DNA fragments aregenerated and purified using conventional techniques, some of whichinclude restriction enzyme digestion, agarose gel electrophoresis, gelpurification of specific fragments, anion-exchange purification andethanol precipitation. The resulting purified centromere and vectorfragments are enzymatically joined in vitro, using for example T4 DNAligase. The ends of the fragments can be cohesive, as the result ofdigestion with compatible restriction endonucleases or from the additionof compatible oligonucleotide linkers; alternatively the ends of thefragments can be blunt and can be directly joined. Following ligation,the resulting mini chromosomes are introduced into E. coli, otherbacteria, yeast, or plant cells using chemical or physicaltransformation methods. The structure of the resulting mini chromosomescan be determined by recovering them from the host organism andassessing DNA fragment size and composition.

2) Transfer of Mini Chromosome Vector Sequences into Centromere Clonesby Site-specific Recombination:

The mini chromosome vector sequences can be constructed to includesite-specific recombination sequences (for example those recognized bythe bacteriophage P1 Cre recombinase, or the bacteriophage lambdaintegrase, or similar recombination enzymes). A compatible recombinationsite, or a pair of such sites, can also be included in the centromereclones. Incubation of the mini chromosome vector and the centromereclone in the presence of the recombinase enzyme causes strand exchangeto occur between the recombination sites in the two plasmids; theresulting mini chromosomes contain centromere sequences as well as minichromosome vector sequences (FIG. 5). Introducing the DNA moleculesformed in such recombination reactions into E. coli, other bacteria,yeast or plant cells can be followed by selection for marker genespresent on both parental plasmids, allowing the isolation of minichromosomes.

3) Mini Chromosome Vector Tailing Method for Mini ChromosomeConstruction:

Centromere DNA fragments isolated from genomic DNA or from centromereclones can be modified on their ends by treatment with restrictionendonucleases, or by ligation with DNA molecules including, but notlimited to, oligonucleotide linkers, or by the addition of nucleotides,to produce a desired cohesive or blunt end. These fragments aresize-fractionated by, agarose gel electrophoresis or other methods, andthe centromere fragments purified using conventional techniques. Minichromosome vector fragments are generated and purified in a similarmanner, resulting in linear mini chromosome vector sequences with DNAends compatible with those on the centromere fragments. Compatible endsin this case are defined by ends that can be joined in vitro by theaction of a ligase enzyme. As shown in FIG. 6, the two fragments arethen mixed so that the mini chromosome vector molecules are present inat least two-fold molar excess over the centromere fragments. Thefragments are joined by the addition of a ligase enzyme (for examplebacteriophage T4 DNA ligase), resulting in the formation of DNAmolecules in which mini chromosome vector molecules have been joined toboth ends of the same centromere fragment. Digestion of the ligationmixture with a rare-cutting restriction or homing endonuclease (forexample endonucleases with recognition sequences of 8 or more bases)results in linear mini chromosome precursors consisting of a fragment ofthe original mini chromosome vector attached to each end of thecentromere fragment. The ends of this hybrid molecule are compatiblebecause they were created by the same restriction enzyme. This linearmini chromosome precursor is purified, for example, by agarose gelelectrophoresis followed by gel purification of the DNA fragments of theexpected length. The purified DNA molecules are circularized by joiningthe ends, for example by treatment with a DNA ligase enzyme. Theresulting mini chromosome molecules can be introduced into E. coli,other bacteria, yeast or plant cells, followed by purification andcharacterization using conventional methods.

VII. Use of Mini Chromosomes for Plant Transformation

1) Delivery of Mini Chromosomes into Plant Cells:

Mini chromosomes are purified and delivered into plant cells, eitherindividually or as a mixture. The mini chromosomes can be eithercircular or linear or mixtures thereof. The plant cells used for minichromosome delivery can be either intact seedlings, immature or matureplants, parts of seedlings or plants, specific plant tissues (forexample leaves, stems, roots, flowers, fruits), differentiated tissuescultured in vitro (for example roots), or undifferentiated cells (forexample callus) cultured in vitro. The mini chromosome DNA can bedelivered into plant cells by a variety of methods including but notlimited to the following: electroporation; Agrobacterium-mediated DNAdelivery; virus-mediated DNA delivery; delivery mediated by salts orlipids that facilitate the cellular uptake of DNA; microinjection ofDNA, manipulation into a cell of DNA-coated or DNA-containing particles,droplets, micelles, microspheres, or chemical complexes using a varietyof techniques, including biolistic particle bombardment, opticaltweezers, particle beams, and electrospray apparatus; manipulation ofDNA-coated magnetic particles into the cells by magnetic fields; DNAdelivery into cells by cell wounding using micro-needles (for examplesilicon carbide needles); sonication or other acoustic treatment of thecells to facilitate DNA uptake; fusion of plant cells with other celltypes carrying a mini chromosome, including bacterial, yeast, or otherplant cells; any other electrical, chemical, physical, or biologicalmechanism that results in the introduction of mini chromosome DNA intothe plant cell

2) Isolating Plant Cells Containing Mini Chromosomes:

Following mini chromosome delivery, plant cells, plant tissues, orcomplete plants carrying the mini chromosome can be isolated by avariety of selection methods. Selection involves subjecting the plantcells, tissues or plants to chemical, environmental, or mechanicaltreatments that enrich for those cells, tissue or plants that contain amini chromosome. The selection methods include but are not limited to:fluorescence-activated cell sorting of cells, cell clumps, or cellprotoplasts based on expression of a marker protein encoded by the minichromosome. (for example, a fluorescent protein such as DsRed); affinitypurification of cells, cell clumps, or protoplasts based on expressionof a cell wall protein, membrane protein, or membrane-associated proteinencoded by the mini chromosome; any cell fractionation method capable ofseparating cells based on their density, size or shape to enrich forcells with a property that differs from that of the starting populationand is conferred by the mini chromosome; selection of cells forresistance to an antibiotic conferred by the mini chromosome; selectionof cells for resistance to an herbicide conferred by the minichromosome; selection of cells for resistance to a toxic metal, salt,mineral or other substance conferred by the mini chromosome; selectionof cells for resistance to abiotic stress (for example heat, cold, acid,base, osmotic stress) conferred by the mini chromosome; selection ofcells capable of utilizing a carbon source or other nutrient source notnormally utilized by plant cells, this utilization function beingconferred by the mini chromosome. As a result of the treatment, apopulation of plant cells can be obtained that contain mini chromosomes.Individual clones or sub-populations of these cells can be expanded inculture for further characterization.

Alternatively, plant cells, plant tissues, or complete plants that carrymini chromosomes can be identified by direct screening. Such methodsinvolve subjecting each cell, plant, or tissue to diagnostic testsindicative of the presence of the mini chromosome. These tests caninclude direct assays for the presence of mini chromosome DNA, orindirect assays for properties conferred by the mini chromosome. Directassays for the presence of the mini chromosome DNA include but are notlimited to: staining of cells with DNA-binding molecules to allowdetection of an additional chromosome; in situ hybridization withlabeled DNA probes corresponding to sequences present on the minichromosome; southern blots or dot blots of DNA extracted from the cells,plant or tissue and probed with labeled DNA sequences corresponding tosequences present on the mini chromosome; electrophoresis of genomic DNAextracted from the cells, plant or tissue under conditions that allowidentification of the mini chromosome; amplification of specificsequences present on the mini chromosome from genomic DNA extracted fromthe cells, plant or tissue using the polymerase chain reaction. Indirectassays for properties conferred by the mini chromosome include but arenot limited to: detection of the expression of a fluorescent markerencoded by the mini chromosome by fluorescence microscopy, flowcytometery or fluorimetry; detection of the expression of a proteinencoded by the mini chromosome by use of specific antibodies, or anyother reagent capable of specifically binding to the protein; use ofcell fractionation methods capable of detecting a specific density, sizeor shape of the cells or tissues, that is conferred by the minichromosome; growth of cells, seedlings, plants or tissues on anantibiotic-containing medium to determine the presence of anantibiotic-resistance gene encoded by the mini chromosome; growth ofcells, seedlings, plants or tissues on an herbicide-containing medium todetermine the presence of an herbicide-resistance gene encoded by themini chromosome; growth of cells, seedlings, plants or tissues on amedium containing a toxic metal, salt, mineral or other substance todetermine the presence of an gene conferring resistance to thissubstance encoded by the mini chromosome; growth of cells, tissues orplants under conditions of abiotic stress (for example heat, cold, acid,base, osmotic stress) to determine the presence of a gene conferringresistance to this stress encoded by the mini chromosome; growth ofcells on a medium containing a carbon source or other nutrient sourcenormally not utilized by plant cells, to determine the presence of autilization function conferred by the mini chromosome.

3) Characterization of Plant Cell Clones Containing Mini Chromosomes

Plant cells, tissues, or entire plants containing mini chromosomes canbe further characterized to determine whether the mini chromosome is anautonomous DNA molecule, or whether it is associated with one of theplant cell's chromosomes by integration. The methods used for thisanalysis include, but are not limited to, the following:

-   1) Detection of marker protein expression by microscopy, flow    cytometry, fluorimetry, enzymatic assays, cell staining or any other    technique that allows the detection of a marker protein having a    specific enzymatic activity, or conferring a specific color, or    fluorescence property onto the cells. For example, if a cell line    has been selected for containing a mini chromosome by selecting for    the function of a resistance gene encoded by the mini chromosome,    and if a marker protein is also encoded by the mini chromosome, then    expression of this marker protein in the selected cells is an    indication of the presence of the entire mini chromosome, and could    indicate autonomy of this mini chromosome from the cell's other    chromosomes.-   2) Use of gel electrophoresis to detect a mini chromosome in genomic    DNA isolated from the plant cells, tissue or entire plants. For    example, genomic DNA isolated from the cells, tissues or plants can    be fractionated by gel electrophoresis, either intact or following    digestion with restriction endonucleases or homing endonucleases,    allowing the detection of a mini chromosome or a fragment of a mini    chromosome.-   3) Use of southern blots or dot blots of DNA extracted from the    cells, tissue or plants to detect the presence of specific sequences    contained on the mini chromosome. For example, digestion of genomic    DNA extracted from the cells, tissues or plants can be fractionated    by agarose gel electrophoresis, blotted onto a DNA-binding membrane,    and probed with labeled DNA sequences corresponding to sequences    present on the mini chromosome to detect specific fragments of mini    chromosome DNA, and thus allowing the determination of the    autonomous, or integrated structure of the mini chromosome.-   4) Cytological techniques for directly visualizing the mini    chromosome in the transformed cells, such as staining of cells with    DNA-binding dyes or in situ hybridization with labeled DNA probes    corresponding to sequences present on the mini chromosome.-   5) Genetic analysis of marker segregation by scoring marker    inheritance in progeny of a plant containing a mini chromosome. For    example, markers present on an autonomous mini chromosome will    segregate independently from markers on the arms of the host    chromosomes in a population of F2 progeny generated from a cross    between a line carrying a mini chromosome and a second marked line    that doesn't carry the mini chromosome. Markers include but are not    limited to: visible markers conferring a visible characteristic to    the plant; selectable markers, conferring resistance to an    antibiotic, herbicide, or other toxic compound; enzymatic markers,    conferring an enzymatic activity that can be assays in the plant or    in extracts made from the plant; protein markers, allowing the    specific detection of a protein expressed in the plant; molecular    markers, such as restriction fragment length polymorphisms,    amplified fragment length polymorphisms, short sequence repeat    (microsatellite) markers, presence of certain sequences in the DNA    of the plant as detected by the polymerase chain reaction, single    nucleotide polymorphisms or cleavable amplified polymorphic sites.    4) Plant Regeneration from Transformed Cell Clones:

Plant cells or tissues that harbor mini chromosomes can be used toregenerate entire plants. This will be accomplished with standardtechniques of plant regeneration from differentiated tissues orundifferentiated cells. Typically, transformed tissues or callus aresubjected to a series of treatments with media containing variousmixtures of plant hormones and growth regulators that promote theformation of a plant embryo, specific plant tissues or organs, or acomplete plant (roots and shoot) from the starting cells or tissues.Following plant regeneration, the plant can be grown either in sterilemedia or in soil.

VIII. Testing Mini Chromosome Inheritance in Plant Cells

The inheritance of mini chromosomes can be measured through one or morecell divisions. After isolating cells, tissues, or entire plants thatcontain the mini chromosome, the population of cells is allowed to grow(either with or with out selection), and the presence of the minichromosome is monitored as the cells divide. Mini chromosomes can bedetected in cells by a variety of methods, including but not limited to:detection of fluorescence or any other visual characteristic arisingfrom a marker protein gene present on the mini chromosome; resistance toan antibiotic, herbicide, toxic metal, salt, mineral or other substance,or abiotic stress as outlined above (Isolating plant cells containingmini chromosomes); staining of cells with DNA-binding molecules to allowdetection of an additional chromosome; in situ hybridization withlabeled DNA probes corresponding to sequences present on the minichromosome; southern blots or dot blots of DNA extracted from the cellpopulation and probed with labeled DNA sequences corresponding tosequences present on the mini chromosome; expression of a marker enzymeencoded by a gene present on the mini chromosome (i.e. luciferase,alkaline phosphatase, beta-galactosidase, etc.) that can be assayed inthe cells or in an extract made from the cells.

The percentage of cells containing the chromosome is determined atregular intervals during this growth phase. The change in the fractionof cells harboring the mini chromosome, divided by the number of celldivisions, represents the average mini chromosome loss rate. Minichromosomes with the lowest loss rates have the highest level ofinheritance.

IX. Recovery of Mini Chromosomes from Plant Cells

Recovery of mini chromosomes from plant cells can be achieved by avariety of techniques, including, but not limited to, the following:

-   1) Extracting the genomic DNA of transformed plant cells and    introducing that DNA into E. coli, other bacteria or yeast and    selecting for the antibiotic resistance genes present on the mini    chromosome.-   2) Isolation of chromosomes from cells, tissues or plants containing    mini chromosomes, and sorting these by flow cytometry to allow the    separation of chromosomes of different size;-   3) Isolation of individual chromosomes from a cell harboring mini    chromosomes by micro-manipulation involving mechanical devices such    as needles made of glass, metal or other suitable substances, or    other techniques such as optical tweezers, or micro-suction devices.-   4) Combinations of the above, for example chromosome isolation by    flow cytometry or micromanipulation followed by introduction into E.    coli, other bacteria, yeast or plant cells.

The resulting mini chromosomes “rescued” in this fashion may differ fromtheir parental molecules in total size, size of the centromere, presenceor absence of additional sequences, and overall arrangement of thesequences. These procedures allow the isolation of DNA molecules capableof replicating and segregating in plant cells without having to testmini chromosomes individually. For example, after delivery of pools ofmini chromosomes, or pools of centromere clones into plant cells,tissues or whole plants, and recovering them by the methods listedabove, facilitates the selection of specific mini chromosomes orcentromere clones that remain autonomous in plant cells. Whereas planttransformation with mini chromosomes relies on the sequences contributedby mini chromosome vectors, the recovery methods do not necessarilyrequire mini chromosome vector sequences; as a result, pools ofcentromere clones can be delivered into plant cells followed by recoveryof the ones that replicated and persist.

X. Exogenous Genes for Expression in Plants

One particularly important advance of the present invention is that itprovides methods and compositions for expression of exogenous genes inplant cells. One advance of the constructs of the current invention isthat they enable the introduction of multiple genes (often referred toas gene “stacking”), potentially representing an entire biochemicalpathway, or any combination of genes encoding different biochemicalprocesses or pathways. Significantly, the current invention allows forthe transformation of plant cells with a mini chromosome comprising anumber of structural genes. Another advantage is that more than one minichromosome could be introduced, allowing combinations of genes to bemoved and shuffled. Moreover, the ability to eliminate a mini chromosomefrom a plant would provide additional flexibility, making it possible toalter the set of genes contained within a plant. Further, by usingsite-specific recombinases, it should be possible to add genes to anexisting mini chromosome once it is in a plant.

Added genes often will be genes that direct the expression of aparticular protein or polypeptide product, but they also may benon-expressible DNA segments, e.g., transposons such as Ds that do notdirect their own transposition. As used herein, an “expressible gene” isany gene that is capable of being transcribed into RNA (e.g., mRNA,antisense RNA, etc.) or translated into a protein, expressed as a traitof interest, or the like, etc., and is not limited to selectable,screenable or non-selectable marker-genes. The inventors alsocontemplate that, where both an expressible gene that is not necessarilya marker gene is employed in combination with a marker gene, one mayemploy the separate genes on either the same or different DNA segmentsfor transformation. In the latter case, the different vectors may bedelivered concurrently to recipient cells to maximize cotransformationor may be delivered sequentially.

The choice of the particular DNA segments to be delivered to therecipient cells often will depend on the purpose of the transformation.One of the major purposes of transformation of crop plants is to addsome commercially desirable, agronomically important traits to theplant. Such traits include, but are not limited to, herbicide resistanceor tolerance; insect resistance or tolerance; disease resistance ortolerance (viral, bacterial, fungal, nematode); stress tolerance and/orresistance, as exemplified by resistance or tolerance to drought, heat,chilling, freezing, excessive moisture, salt stress; oxidative stress;increased yields; food content and makeup; physical appearance; malesterility; drydown; standability; prolificacy; starch quantity andquality; oil quantity and quality; protein quality and quantity; aminoacid composition; the production of a pharmaceutically active protein;the production of a small molecule with medicinal properties; theproduction of a chemical including those with industrial utility; theproduction of nutraceuticals, carbohydrates, RNAs, lipids, fuels, dyes,pigments, vitamins, scents, flavors, vaccines, antibodies, hormones, andthe like. Additionally one could create a library of an entire genomefrom any organism or organelle including mammals, plants, microbes,fungi, bacteria, represented on mini chromosomes. Furthermore one couldincorporate a desired genomic segment such as one that includes aquantitative trait onto a mini chromosome. One may desire to incorporateone or more genes conferring any such desirable trait or traits, suchas, for example, a gene or genes encoding herbicide resistance.

In certain embodiments, the present invention contemplates thetransformation of a recipient cell with mini chromosomes comprising morethan one exogenous gene. An “exogenous gene,” can be a gene not normallyfound in the host genome in an identical context, or alternatively, themini chromosome could be used to introduce extra copies of host genesinto a cell. The gene may be isolated from a different species than thatof the host genome, or alternatively, isolated from the host genome butoperably linked to one or more regulatory regions which differ fromthose found in the unaltered, native gene. Two or more exogenous genesalso can be supplied in a single transformation event using eitherdistinct transgene-encoding vectors, or using a single vectorincorporating two or more gene coding sequences. For example, plasmidsbearing the bar and aroA expression units in either convergent,divergent, or colinear orientation, are considered to be particularlyuseful. Further preferred combinations are those of an insect resistancegene, such as a Bt gene, along with a protease inhibitor gene such aspinII, or the use of bar in combination with either of the above genes.Of course, any two or more transgenes of any description, such as thoseconferring herbicide, insect, disease (viral, bacterial, fungal,nematode) or drought resistance, male sterility, drydown, standability,prolificacy, starch properties, oil quantity and quality, modifiedchemical production, pharmaceutical or nutraceutical properties,bioremediation properties, increased biomass, altered growth rate,altered fitness, altered salinity tolerance, altered thermal tolerance,altered growth form, altered composition, altered metabolism, alteredbiodegradability, altered CO₂ fixation, altered stress tolerance,presence of bioindicator activity, altered digestibility by humans oranimals, altered allergenicity, altered mating characteristics, alteredpollen dispersal, altered appearance, improved environmental impact,nitrogen fixation capability, or those increasing yield or nutritionalquality may be employed as desired.

(i) Herbicide Resistance

The genes encoding phosphinothricin acetyltransferase (bar and pat),glyphosate tolerant EPSP synthase genes, the glyphosate degradativeenzyme gene gox encoding glyphosate oxidoreductase, deh (encoding adehalogenase enzyme that inactivates dalapon), herbicide resistant(e.g., sulfonylurea and imidazolinone) acetolactate synthase, and bxngenes (encoding a nitrilase enzyme that degrades bromoxynil) are goodexamples of herbicide resistant genes for use in transformation. The barand pat genes code for an enzyme, phosphinothricin acetyltransferase(PAT), which inactivates the herbicide phosphinothricin and preventsthis compound from inhibiting glutamine synthetase enzymes. The enzyme5-enolpyruvylshikimate 3-phosphate synthase (EPSP Synthase), is normallyinhibited by the herbicide N-(phosphonomethyl)glycine (glyphosate).However, genes are known that encode glyphosate-resistant EPSP synthaseenzymes. These genes are particularly contemplated for use in planttransformation. The deh gene encodes the enzyme dalapon dehalogenase andconfers resistance to the herbicide dalapon. The bxn gene codes for aspecific nitrilase enzyme that converts bromoxynil to a non-herbicidaldegradation product.

(ii) Insect Resistance

Potential insect resistance genes that can be introduced includeBacillus thuringiensis crystal toxin genes or Bt genes (Watrud et al.,1985). Bt genes may provide resistance to lepidopteran or coleopteranpests such as European Corn Borer (ECB). Preferred Bt toxin genes foruse in such embodiments include the CryIA(b) and CryIA(c) genes.Endotoxin genes from other species of B. thuringiensis which affectinsect growth or development also may be employed in this regard.

It is contemplated that preferred Bt genes for use in the transformationprotocols disclosed herein will be those in which the coding sequencehas been modified to effect increased expression in plants, and moreparticularly, in monocot plants. Means for preparing synthetic genes arewell known in the art and are disclosed in, for example, U.S. Pat. Nos.5,500,365 and 5,689,052, each of the disclosures of which arespecifically incorporated herein by reference in their entirety.Examples of such modified Bt toxin genes include a synthetic Bt CryIA(b)gene (Perlak et al., 1991), and the synthetic CryIA(c) gene termed 1800b(PCT Application WO 95/06128). Some examples of other Bt toxin genesknown to those of skill in the art are given in Table 1 below.

TABLE 1 Bacillus thuringiensis δ-Endotoxin Genes^(a) New Old GenBankNomenclature Nomenclature Accession Cry1Aa CryIA(a) M11250 Cry1AbCryIA(b) M13898 Cry1Ac CryIA(c) M11068 Cry1Ad CryIA(d) M73250 Cry1AeCryIA(e) M65252 Cry1Ba CryIB X06711 Cry1Bb ET5 L32020 Cry1Bc PEG5 Z46442Cry1Bd CryE1 U70726 Cry1Ca CryIC X07518 Cry1Cb CryIC(b) M97880 Cry1DaCryID X54160 Cry1Db PrtB Z22511 Cry1Ea CryIE X53985 Cry1Eb CryIE(b)M73253 Cry1Fa CryIF M63897 Cry1Fb PrtD Z22512 Cry1Ga PrtA Z22510 Cry1GbCryH2 U70725 Cry1Ha PrtC Z22513 Cry1Hb U35780 Cry1Ia CryV X62821 Cry1IbCryV U07642 Cry1Ja ET4 L32019 Cry1Jb ET1 U31527 Cry1K U28801 Cry2AaCryIIA M31738 Cry2Ab CryIIB M23724 Cry2Ac CryIIC X57252 Cry3A CryIIIAM22472 Cry3Ba CryIIIB X17123 Cry3Bb CryIIIB2 M89794 Cry3C CryIIID X59797Cry4A CryIVA Y00423 Cry4B CryIVB X07423 Cry5Aa CryVA(a) L07025 Cry5AbCryVA(b) L07026 Cry6A CryVIA L07022 Cry6B CryVIB L07024 Cry7Aa CryIIICM64478 Cry7Ab CryIIICb U04367 Cry8A CryIIIE U04364 Cry8B CryIIIG U04365Cry8C CryIIIF U04366 Cry9A CryIG X58120 Cry9B CryIX X75019 Cry9C CryIHZ37527 Cry10A CryIVC M12662 Cry11A CryIVD M31737 Cry11B Jeg80 X86902Cry12A CryVB L07027 Cry13A CryVC L07023 Cry14A CryVD U13955 Cry15A 34kDa M76442 Cry16A cbm71 X94146 Cry17A cbm71 X99478 Cry18A CryBP1 X99049Cry19A Jeg65 Y08920 Cyt1Aa CytA X03182 Cyt1Ab CytM X98793 Cyt2A CytBZ14147 Cyt2B CytB U52043 ^(a)Adapted from:http://epunix.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.html

Protease inhibitors also may provide insect resistance (Johnson et al.,1989), and will thus have utility in plant transformation. The use of aprotease inhibitor II gene, pinII, from tomato or potato is envisionedto be particularly useful. Even more advantageous is the use of a pinIIgene in combination with a Bt toxin gene, the combined effect of whichhas been discovered to produce synergistic insecticidal activity. Othergenes which encode inhibitors of the insect's digestive system, or thosethat encode enzymes or co-factors that facilitate the production ofinhibitors, also may be useful. This group may be exemplified byoryzacystatin and amylase inhibitors such as those from wheat andbarley.

Also, genes encoding lectins may confer additional or alternativeinsecticide properties. Lectins (originally termed phytohemagglutinins)are multivalent carbohydrate-binding proteins which have the ability toagglutinate red blood cells from a range of species. Lectins have beenidentified recently as insecticidal agents with activity againstweevils, ECB and rootworm (Murdock et al., 1990; Czapla & Lang, 1990).Lectin genes contemplated to be useful include, for example, barley andwheat germ agglutinin (WGA) and rice lectins (Gatehouse et al., 1984),with WGA being preferred.

Genes controlling the production of large or small polypeptides activeagainst insects when introduced into the insect pests, such as, e.g.,lytic peptides, peptide hormones and toxins and venoms, form anotheraspect of the invention. For example, it is contemplated that theexpression of juvenile hormone esterase, directed towards specificinsect pests, also may result in insecticidal activity, or perhaps causecessation of metamorphosis (Hammock et al., 1990).

Transgenic plants expressing genes which encode enzymes that affect theintegrity of the insect cuticle form yet another aspect of theinvention. Such genes include those encoding, e.g., chitinase,proteases, lipases and also genes for the production of nikkomycin, acompound that inhibits chitin synthesis, the introduction of any ofwhich is contemplated to produce insect resistant plants. Genes thatcode for activities that affect insect molting, such as those affectingthe production of ecdysteroid UDP-glucosyl transferase, also fall withinthe scope of the useful transgenes of the present invention.

Genes that code for enzymes that facilitate the production of compoundsthat reduce the nutritional quality of the host plant to insect pestsalso are encompassed by the present invention. It may be possible, forinstance, to confer insecticidal activity on a plant by altering itssterol composition. Sterols are obtained by insects from their diet andare used for hormone synthesis and membrane stability. Thereforealterations in plant sterol composition by expression of novel genes,e.g., those that directly promote the production of undesirable sterolsor those that convert desirable sterols into undesirable forms, couldhave a negative effect on insect growth and/or development and henceendow the plant with insecticidal activity. Lipoxygenases are naturallyoccurring plant enzymes that have been shown to exhibit anti-nutritionaleffects on insects and to reduce the nutritional quality of their diet.Therefore, further embodiments of the invention concern transgenicplants with enhanced lipoxygenase activity which may be resistant toinsect feeding.

Tripsacum dactyloides is a species of grass that is resistant to certaininsects, including corn root worm. It is anticipated that genes encodingproteins that are toxic to insects or are involved in the biosynthesisof compounds toxic to insects will be isolated from Tripsacum and thatthese novel genes will be useful in conferring resistance to insects. Itis known that the basis of insect resistance in Tripsacum is genetic,because said resistance has been transferred to Zea mays via sexualcrosses (Branson and Guss, 1972). It is further anticipated that othercereal, monocot or dicot plant species may have genes encoding proteinsthat are toxic to insects which would be useful for producing insectresistant plants.

Further genes encoding proteins characterized as having potentialinsecticidal activity also may be used as transgenes in accordanceherewith. Such genes include, for example, the cowpea trypsin inhibitor(CpTI; Hilder et al., 1987) which may be used as a rootworm deterrent;genes encoding avermectin (Avermectin and Abamectin., Campbell, W. C.,Ed., 1989; Ikeda et al., 1987) which may prove particularly useful as acorn rootworm deterrent; ribosome inactivating protein genes; and evengenes that regulate plant structures. Transgenic plants includinganti-insect antibody genes and genes that code for enzymes that canconvert a non-toxic insecticide (pro-insecticide) applied to the outsideof the plant into an insecticide inside the plant also are contemplated.

(iii) Environment or Stress Resistance

Improvement of a plants ability to tolerate various environmentalstresses such as, but not limited to, drought, excess moisture,chilling, freezing, high temperature, salt, and oxidative stress, alsocan be effected through expression of novel genes. It is proposed thatbenefits may be realized in terms of increased resistance to freezingtemperatures through the introduction of an “antifreeze” protein such asthat of the Winter Flounder (Cutler et al., 1989) or synthetic genederivatives thereof. Improved chilling tolerance also may be conferredthrough increased expression of glycerol-3-phosphate acetyltransferasein chloroplasts (Wolter et al., 1992). Resistance to oxidative stress(often exacerbated by conditions such as chilling temperatures incombination with high light intensities) can be conferred by expressionof superoxide dismutase (Gupta et al., 1993), and may be improved byglutathione reductase (Bowler et al., 1992). Such strategies may allowfor tolerance to freezing in newly emerged fields as well as extendinglater maturity higher yielding varieties to earlier relative maturityzones.

It is contemplated that the expression of novel genes that favorablyeffect plant water content, total water potential, osmotic potential,and turgor will enhance the ability of the plant to tolerate drought. Asused herein, the terms “drought resistance” and “drought tolerance” areused to refer to a plants increased resistance or tolerance to stressinduced by a reduction in water availability, as compared to normalcircumstances, and the ability of the plant to function and survive inlower-water environments. In this aspect of the invention it isproposed, for example, that the expression of genes encoding for thebiosynthesis of osmotically-active solutes, such as polyol compounds,may impart protection against drought. Within this class are genesencoding for mannitol-L-phosphate dehydrogenase (Lee and Saier, 1982)and trehalose-6-phosphate synthase (Kaasen et al., 1992). Through thesubsequent action of native phosphatases in the cell or by theintroduction and coexpression of a specific phosphatase, theseintroduced genes will result in the accumulation of either mannitol ortrehalose, respectively, both of which have been well documented asprotective compounds able to mitigate the effects of stress. Mannitolaccumulation in transgenic tobacco has been verified and preliminaryresults indicate that plants expressing high levels of this metaboliteare able to tolerate an applied osmotic stress (Tarczynski et al., 1992,1993).

Similarly, the efficacy of other metabolites in protecting either enzymefunction (e.g., alanopine or propionic acid) or membrane integrity(e.g., alanopine) has been documented (Loomis et al., 1989), andtherefore expression of genes encoding for the biosynthesis of thesecompounds might confer drought resistance in a manner similar to orcomplimentary to mannitol. Other examples of naturally occurringmetabolites that are osmotically active and/or provide some directprotective effect during drought and/or desiccation include fructose,erythritol (Coxson et al., 1992), sorbitol, dulcitol (Karsten et al.,1992), glucosylglycerol (Reed et al., 1984; ErdMann et al., 1992),sucrose, stachyose (Koster and Leopold, 1988; Blackman et al., 1992),raffinose (Bernal-Lugo and Leopold, 1992), proline (Rensburg et al.,1993), glycine betaine, ononitol and pinitol (Vernon and Bohnert, 1992).Continued canopy growth and increased reproductive fitness during timesof stress will be augmented by introduction and expression of genes suchas those controlling the osmotically active compounds discussed aboveand other such compounds. Currently preferred genes which promote thesynthesis of an osmotically active polyol compound are genes whichencode the enzymes mannitol-1-phosphate dehydrogenase,trehalose-6-phosphate synthase and myoinositol 0-methyltransferase.

It is contemplated that the expression of specific proteins also mayincrease drought tolerance. Three classes of Late Embryogenic Proteinshave been assigned based on structural similarities (see Dure et al.,1989). All three classes of LEAs have been demonstrated in maturing(i.e. desiccating) seeds. Within these 3 types of LEA proteins, theType-II (dehydrin-type) have generally been implicated in drought and/ordesiccation tolerance in vegetative plant parts (i.e. Mundy and Chua,1988; Piatkowski et al., 1990; Yamaguchi-Shinozaki et al., 1992).Recently, expression of a Type-III LEA (HVA-1) in tobacco was found toinfluence plant height, maturity and drought tolerance (Fitzpatrick,1993). In rice, expression of the HVA-1 gene influenced tolerance towater deficit and salinity (Xu et al., 1996). Expression of structuralgenes from all three LEA groups may therefore confer drought tolerance.Other types of proteins induced during water stress include thiolproteases, aldolases and transmembrane transporters (Guerrero et al.,1990), which may confer various protective and/or repair-type functionsduring drought stress. It also is contemplated that genes that effectlipid biosynthesis and hence membrane composition might also be usefulin conferring drought resistance on the plant.

Many of these genes for improving drought resistance have complementarymodes of action. Thus, it is envisaged that combinations of these genesmight have additive and/or synergistic effects in improving droughtresistance in plants. Many of these genes also improve freezingtolerance (or resistance); the physical stresses incurred duringfreezing and drought are similar in nature and may be mitigated insimilar fashion. Benefit may be conferred via constitutive expression ofthese genes, but the preferred means of expressing these novel genes maybe through the use of a turgor-induced promoter (such as the promotersfor the turgor-induced genes described in Guerrero et al., 1990 andShagan et al., 1993 which are incorporated herein by reference). Spatialand temporal expression patterns of these genes may enable plants tobetter withstand stress.

It is proposed that expression of genes that are involved with specificmorphological traits that allow for increased water extractions fromdrying soil would be of benefit. For example, introduction andexpression of genes that alter root characteristics may enhance wateruptake. It also is contemplated that expression of genes that enhancereproductive fitness during times of stress would be of significantvalue. For example, expression of genes that improve the synchrony ofpollen shed and receptiveness of the female flower parts, i.e., silks,would be of benefit. In addition it is proposed that expression of genesthat minimize kernel abortion during times of stress would increase theamount of grain to be harvested and hence be of value.

Given the overall role of water in determining yield, it is contemplatedthat enabling plants to utilize water more efficiently, through theintroduction and expression of novel genes, will improve overallperformance even when soil water availability is not limiting. Byintroducing genes that improve the ability of plants to maximize waterusage across a full range of stresses relating to water availability,yield stability or consistency of yield performance may be realized.

(iv) Disease Resistance

It is proposed that increased resistance to diseases may be realizedthrough introduction of genes into plants, for example, intomonocotyledonous plants such as maize. It is possible to produceresistance to diseases caused by viruses, bacteria, fungi and nematodes.It also is contemplated that control of mycotoxin producing organismsmay be realized through expression of introduced genes.

Resistance to viruses may be produced through expression of novel genes.For example, it has been demonstrated that expression of a viral coatprotein in a transgenic plant can impart resistance to infection of theplant by that virus and perhaps other closely related viruses (Cuozzo etal., 1988, Hemenway et al., 1988, Abel et al., 1986). It is contemplatedthat expression of antisense genes targeted at essential viral functionsmay also impart resistance to viruses. For example, an antisense genetargeted at the gene responsible for replication of viral nucleic acidmay inhibit replication and lead to resistance to the virus. It isbelieved that interference with other viral functions through the use ofantisense genes also may increase resistance to viruses. Further, it isproposed that it may be possible to achieve resistance to virusesthrough other approaches, including, but not limited to the use ofsatellite viruses.

It is proposed that increased resistance to diseases caused by bacteriaand fungi may be realized through introduction of novel genes. It iscontemplated that genes encoding so-called “peptide antibiotics,”pathogenesis related (PR) proteins, toxin resistance, and proteinsaffecting host-pathogen interactions such as morphologicalcharacteristics will be useful. Peptide antibiotics are polypeptidesequences which are inhibitory to growth of bacteria and othermicroorganisms. For example, the classes of peptides referred to ascecropins and magainins inhibit growth of many species of bacteria andfungi. It is proposed that expression of PR proteins in monocotyledonousplants such as maize may be useful in conferring resistance to bacterialdisease. These genes are induced following pathogen attack on a hostplant and have been divided into at least five classes of proteins (Bol,Linthorst, and Cornelissen, 1990). Included amongst the PR proteins areβ-1,3-glucanases, chitinases, and osmotin and other proteins that arebelieved to function in plant resistance to disease organisms. Othergenes have been identified that have antifungal properties, e.g., UDA(stinging nettle lectin) and hevein (Broakaert et al., 1989;Barkai-Golan et al., 1978). It is known that certain plant diseases arecaused by the production of phytotoxins. It is proposed that resistanceto these diseases would be achieved through expression of a novel genethat encodes an enzyme capable of degrading or otherwise inactivatingthe phytotoxin. It also is contemplated that expression of novel genesthat alter the interactions between the host plant and pathogen may beuseful in reducing the ability of the disease organism to invade thetissues of the host plant, e.g., an increase in the waxiness of the leafcuticle or other morphological characteristics.

(v) Plant Agronomic Characteristics

Two of the factors determining where crop plants can be grown are theaverage daily temperature during the growing season and the length oftime between frosts. Within the areas where it is possible to grow aparticular crop, there are varying limitations on the maximal time it isallowed to grow to maturity and be harvested. For example, a variety tobe grown in a particular area is selected for its ability to mature anddry down to harvestable moisture content within the required period oftime with maximum possible yield. Therefore, crops of varying maturitiesis developed for different growing locations. Apart from the need to drydown sufficiently to permit harvest, it is desirable to have maximaldrying take place in the field to minimize the amount of energy requiredfor additional drying post-harvest. Also, the more readily a productsuch as grain can dry down, the more time there is available for growthand kernel fill. It is considered that genes that influence maturityand/or dry down can be identified and introduced into plant lines usingtransformation techniques to create new varieties adapted to differentgrowing locations or the same growing location, but having improvedyield to moisture ratio at harvest. Expression of genes that areinvolved in regulation of plant development may be especially useful.

It is contemplated that genes may be introduced into plants that wouldimprove standability and other plant growth characteristics. Expressionof novel genes in plants which confer stronger stalks, improved rootsystems, or prevent or reduce ear droppage would be of great value tothe farmer. It is proposed that introduction and expression of genesthat increase the total amount of photoassimilate available by, forexample, increasing light distribution and/or interception would beadvantageous. In addition, the expression of genes that increase theefficiency of photosynthesis and/or the leaf canopy would furtherincrease gains in productivity. It is contemplated that expression of aphytochrome gene in crop plants may be advantageous. Expression of sucha gene may reduce apical dominance, confer semidwarfism on a plant, andincrease shade tolerance (U.S. Pat. No. 5,268,526). Such approacheswould allow for increased plant populations in the field.

(vi) Nutrient Utilization

The ability to utilize available nutrients may be a limiting factor ingrowth of crop plants. It is proposed that it would be possible to alternutrient uptake, tolerate pH extremes, mobilization through the plant,storage pools, and availability for metabolic activities by theintroduction of novel genes. These modifications would allow a plantsuch as maize to more efficiently utilize available nutrients. It iscontemplated that an increase in the activity of, for example, an enzymethat is normally present in the plant and involved in nutrientutilization would increase the availability of a nutrient. An example ofsuch an enzyme would be phytase. It is further contemplated thatenhanced nitrogen utilization by a plant is desirable. Expression of aglutamate dehydrogenase gene in plants, e.g., E. coli gdhA genes, maylead to increased fixation of nitrogen in organic compounds.Furthermore, expression of gdhA in plants may lead to enhancedresistance to the herbicide glufosinate by incorporation of excessammonia into glutamate, thereby detoxifying the ammonia. It also iscontemplated that expression of a novel gene may make a nutrient sourceavailable that was previously not accessible, e.g., an enzyme thatreleases a component of nutrient value from a more complex molecule,perhaps a macromolecule.

(vii) Male Sterility

Male sterility is useful in the production of hybrid seed. It isproposed that male sterility may be produced through expression of novelgenes. For example, it has been shown that expression of genes thatencode proteins that interfere with development of the maleinflorescence and/or gametophyte result in male sterility. Chimericribonuclease genes that express in the anthers of transgenic tobacco andoilseed rape have been demonstrated to lead to male sterility (Marianiet al., 1990).

A number of mutations were discovered in maize that confer cytoplasmicmale sterility. One mutation in particular, referred to as T cytoplasm,also correlates with sensitivity to Southern corn leaf blight. A DNAsequence, designated TURF-13 (Levings, 1990), was identified thatcorrelates with T cytoplasm. It is proposed that it would be possiblethrough the introduction of TURF-13 via transformation, to separate malesterility from disease sensitivity. As it is necessary to be able torestore male fertility for breeding purposes and for grain production,it is proposed that genes encoding restoration of male fertility alsomay be introduced.

(viii) Improved Nutritional Content

Genes may be introduced into plants to improve the nutrient quality orcontent of a particular crop. Introduction of genes that alter thenutrient composition of a crop may greatly enhance the feed or foodvalue. For example, the protein of many grains is suboptimal for feedand food purposes, especially when fed to pigs, poultry, and humans. Theprotein is deficient in several amino acids that are essential in thediet of these species, requiring the addition of supplements to thegrain. Limiting essential amino acids may include lysine, methionine,tryptophan, threonine, valine, arginine, and histidine. Some amino acidsbecome limiting only after corn is supplemented with other inputs forfeed formulations. The levels of these essential amino acids in seedsand grain may be elevated by mechanisms which include, but are notlimited to, the introduction of genes to increase the biosynthesis ofthe amino acids, decrease the degradation of the amino acids, increasethe storage of the amino acids in proteins, or increase transport of theamino acids to the seeds or grain.

The protein composition of a crop may be altered to improve the balanceof amino acids in a variety of ways including elevating expression ofnative proteins, decreasing expression of those with poor composition,changing the composition of native proteins, or introducing genesencoding entirely new proteins possessing superior composition.

The introduction of genes that alter the oil content of a crop plant mayalso be of value. Increases in oil content may result in increases inmetabolizable-energy-content and density of the seeds for use in feedand food. The introduced genes may encode enzymes that remove or reducerate-limitations or regulated steps in fatty acid or lipid biosynthesis.Such genes may include, but are not limited to, those that encodeacetyl-CoA carboxylase, ACP-acyltransferase, β-ketoacyl-ACP synthase,plus other well known fatty acid biosynthetic activities. Otherpossibilities are genes that encode proteins that do not possessenzymatic activity such as acyl carrier protein. Genes may be introducedthat alter the balance of fatty acids present in the oil providing amore healthful or nutritive feedstuff. The introduced DNA also mayencode sequences that block expression of enzymes involved in fatty acidbiosynthesis, altering the proportions of fatty acids present in crops.

Genes may be introduced that enhance the nutritive value of the starchcomponent of crops, for example by increasing the degree of branching,resulting in improved utilization of the starch in livestock by delayingits metabolism. Additionally, other major constituents of a crop may bealtered, including genes that affect a variety of other nutritive,processing, or other quality aspects. For example, pigmentation may beincreased or decreased.

Feed or food crops may also possess sub-optimal quantities of vitamins,antioxidants or other nutraceuticals, requiring supplementation toprovide adequate nutritive value and ideal health value. Introduction ofgenes that enhance vitamin biosynthesis may be envisioned including, forexample, vitamins A, E, B₁₂, choline, and the like. Mineral content mayalso be sub-optimal. Thus genes that affect the accumulation oravailability of compounds containing phosphorus, sulfur, calcium,manganese, zinc, and iron among others would be valuable.

Numerous other examples of improvements of crops may be used with theinvention. The improvements may not necessarily involve grain, but may,for example, improve the value of a crop for silage. Introduction of DNAto accomplish this might include sequences that alter lignin productionsuch as those that result in the “brown midrib” phenotype associatedwith superior feed value for cattle.

In addition to direct improvements in feed or food value, genes also maybe introduced which improve the processing of crops and improve thevalue of the products resulting from the processing. One use of crops ifvia wetmilling. Thus novel genes that increase the efficiency and reducethe cost of such processing, for example by decreasing steeping time,may also find use. Improving the value of wetmilling products mayinclude altering the quantity or quality of starch, oil, corn glutenmeal, or the components of gluten feed. Elevation of starch may beachieved through the identification and elimination of rate limitingsteps in starch biosynthesis or by decreasing levels of the othercomponents of crops resulting in proportional increases in starch.

Oil is another product of wetmilling, the value of which may be improvedby introduction and expression of genes. Oil properties may be alteredto improve its performance in the production and use of cooking oil,shortenings, lubricants or other oil-derived products or improvement ofits health attributes when used in the food-related applications. Novelfatty acids also may be synthesized which upon extraction can serve asstarting materials for chemical syntheses. The changes in oil propertiesmay be achieved by altering the type, level, or lipid arrangement of thefatty acids present in the oil. This in turn may be accomplished by theaddition of genes that encode enzymes that catalyze the synthesis ofnovel fatty acids and the lipids possessing them or by increasing levelsof native fatty acids while possibly reducing levels of precursors.Alternatively, DNA sequences may be introduced which slow or block stepsin fatty acid biosynthesis resulting in the increase in precursor fattyacid intermediates. Genes that might be added include desaturases,epoxidases, hydratases, dehydratases, and other enzymes that catalyzereactions involving fatty acid intermediates. Representative examples ofcatalytic steps that might be blocked include the desaturations fromstearic to oleic acid and oleic to linolenic acid resulting in therespective accumulations of stearic and oleic acids. Another example isthe blockage of elongation steps resulting in the accumulation of C₈ toC₁₂ saturated fatty acids.

(ix) Production or Assimilation of Chemicals or Biologicals

It may further be considered that a transgenic plant prepared inaccordance with the invention may be used for the production ormanufacturing of useful biological compounds that were either notproduced at all, or not produced at the same level, in the corn plantpreviously. Alternatively, plants produced in accordance with theinvention may be made to metabolize or absorb and concentrate certaincompounds, such as hazardous wastes, thereby allowing bioremediation ofthese compounds.

The novel plants producing these compounds are made possible by theintroduction and expression of one or potentially many genes with theconstructs provided by the invention. The vast array of possibilitiesinclude but are not limited to any biological compound which ispresently produced by any organism such as proteins, nucleic acids,primary and intermediary metabolites, carbohydrate polymers, enzymes foruses in bioremediation, enzymes for modifying pathways that producesecondary plant metabolites such as flavonoids or vitamins, enzymes thatcould produce pharmaceuticals, and for introducing enzymes that couldproduce compounds of interest to the manufacturing industry such asspecialty chemicals and plastics. The compounds may be produced by theplant, extracted upon harvest and/or processing, and used for anypresently recognized useful purpose such as pharmaceuticals, fragrances,and industrial enzymes to name a few.

(x) Non-Protein-Expressing Sequences

DNA may be introduced into plants for the purpose of expressing RNAtranscripts that function to affect plant phenotype yet are nottranslated into protein. Two examples are antisense RNA and RNA withribozyme activity. Both may serve possible functions in reducing oreliminating expression of native or introduced plant genes. However, asdetailed below, DNA need not be expressed to effect the phenotype of aplant.

1. Antisense RNA

Genes may be constructed or isolated, which when transcribed, produceantisense RNA that is complementary to all or part(s) of a targetedmessenger RNA(s). The antisense RNA reduces production of thepolypeptide product of the messenger RNA. Genes may also be constructedto produce double-stranded RNA molecules complementary to all or part ofthe targeted messenger RNA(s). Genes designed in this manner will bereferred to as RNAi constructs; the double-stranded RNA or RNAiconstructs can trigger the sequence-specific degradation of the targetmessenger RNA. The polypeptide product of the target messenger RNA maybe any protein. The aforementioned genes will be referred to asantisense genes and RNAi constructs, respectively. An antisense gene orRNAi construct may thus be introduced into a plant by transformationmethods to produce a novel transgenic plant with reduced expression of aselected protein of interest. For example, the protein may be an enzymethat catalyzes a reaction in the plant. Reduction of the enzyme activitymay reduce or eliminate products of the reaction which include anyenzymatically synthesized compound in the plant such as fatty acids,amino acids, carbohydrates, nucleic acids and the like. Alternatively,the protein may be a storage protein, such as a zein, or a structuralprotein, the decreased expression of which may lead to changes in seedamino acid composition or plant morphological changes respectively. Thepossibilities cited above are provided only by way of example and do notrepresent the full range of applications.

2. Ribozymes

Genes also may be constructed or isolated, which when transcribed,produce RNA enzymes (ribozymes) which can act as endoribonucleases andcatalyze the cleavage of RNA molecules with selected sequences. Thecleavage of selected messenger RNAs can result in the reduced productionof their encoded polypeptide products. These genes may be used toprepare novel transgenic plants which possess them. The transgenicplants may possess reduced levels of polypeptides including, but notlimited to, the polypeptides cited above.

Ribozymes are RNA-protein complexes that cleave nucleic acids in asite-specific fashion. Ribozymes have specific catalytic domains thatpossess endonuclease activity (Kim and Cech, 1987; Gerlach et al., 1987;Forster and Symons, 1987). For example, a large number of ribozymesaccelerate phosphoester transfer reactions with a high degree ofspecificity, often cleaving only one of several phosphoesters in anoligonucleotide substrate (Cech et al., 1981; Michel and Westhof, 1990;Reinhold-Hurek and Shub, 1992). This specificity has been attributed tothe requirement that the substrate bind via specific base-pairinginteractions to the internal guide sequence (“IGS”) of the ribozymeprior to chemical reaction.

Ribozyme catalysis has primarily been observed as part ofsequence-specific cleavage/ligation reactions involving nucleic acids(Joyce, 1989; Cech et al., 1981). For example, U.S. Pat. No. 5,354,855reports that certain ribozymes can act as endonucleases with a sequencespecificity greater than that of known ribonucleases and approachingthat of the DNA restriction enzymes.

Several different ribozyme motifs have been described with RNA cleavageactivity (Symons, 1992). Examples include sequences from the Group Iself splicing introns including Tobacco Ringspot Virus (Prody et al.,1986), Avocado Sunblotch Viroid (Palukaitis et al., 1979; Symons, 1981),and Lucerne Transient Streak Virus (Forster and Symons, 1987). Sequencesfrom these and related viruses are referred to as hammerhead ribozymebased on a predicted folded secondary structure.

Other suitable ribozymes include sequences from RNase P with RNAcleavage activity (Yuan et al., 1992, Yuan and Altman, 1994, U.S. Pat.Nos. 5,168,053 and 5,624,824), hairpin ribozyme structures(Berzal-Herranz et al., 1992; Chowrira et al., 1993) and Hepatitis Deltavirus based ribozymes (U.S. Pat. No. 5,625,047). The general design andoptimization of ribozyme directed RNA cleavage activity has beendiscussed in detail (Haseloff and Gerlach, 1988, Symons, 1992, Chowriraet al., 1994; Thompson et al., 1995).

The other variable on ribozyme design is the selection of a cleavagesite on a given target RNA. Ribozymes are targeted to a given sequenceby virtue of annealing to a site by complimentary base pairinteractions. Two stretches of homology are required for this targeting.These stretches of homologous sequences flank the catalytic ribozymestructure defined above. Each stretch of homologous sequence can vary inlength from 7 to 15 nucleotides. The only requirement for defining thehomologous sequences is that, on the target RNA, they are separated by aspecific sequence which is the cleavage site. For hammerhead ribozyme,the cleavage site is a dinucleotide sequence on the target RNA is auracil (U) followed by either an adenine, cytosine or uracil (A, C or U)(Perriman et al., 1992; Thompson et al., 1995). The frequency of thisdinucleotide occurring in any given RNA is statistically 3 out of 16.Therefore, for a given target messenger RNA of 1,000 bases, 187dinucleotide cleavage sites are statistically possible.

Designing and testing ribozymes for efficient cleavage of a target RNAis a process well known to those skilled in the art. Examples ofscientific methods for designing and testing ribozymes are described byChowrira et al., (1994) and Lieber and Strauss (1995), each incorporatedby reference. The identification of operative and preferred sequencesfor use in down regulating a given gene is simply a matter of preparingand testing a given sequence, and is a routinely practiced “screening”method known to those of skill in the art.

3. Induction of Gene Silencing

It also is possible that genes may be introduced to produce noveltransgenic plants which have reduced expression of a native gene productby the mechanism of co-suppression. It has been demonstrated in tobacco,tomato, and petunia (Goring et al., 1991; Smith et al., 1990; Napoli etal., 1990; van der Krol et al., 1990) that expression of the sensetranscript of a native gene will reduce or eliminate expression of thenative gene in a manner similar to that observed for antisense genes.The introduced gene may encode all or part of the targeted nativeprotein but its translation may not be required for reduction of levelsof that native protein.

4. Non-RNA-Expressing Sequences

DNA elements including those of transposable elements such as Ds, Ac, orMu, may be inserted into a gene to cause mutations. These DNA elementsmay be inserted in order to inactivate (or activate) a gene and thereby“tag” a particular trait. In this instance the transposable element doesnot cause instability of the tagged mutation, because the utility of theelement does not depend on its ability to move in the genome. Once adesired trait is tagged, the introduced DNA sequence may be used toclone the corresponding gene, e.g., using the introduced DNA sequence asa PCR primer together with PCR gene cloning techniques (Shapiro, 1983;Dellaporta et al., 1988). Once identified, the entire gene(s) for theparticular trait, including control or regulatory regions where desired,may be isolated, cloned and manipulated as desired. The utility of DNAelements introduced into an organism for purposes of gene tagging isindependent of the DNA sequence and does not depend on any biologicalactivity of the DNA sequence, i.e., transcription into RNA ortranslation into protein. The sole function of the DNA element is todisrupt the DNA sequence of a gene.

It is contemplated that unexpressed DNA sequences, including novelsynthetic sequences, could be introduced into cells as proprietary“labels” of those cells and plants and seeds thereof. It would not benecessary for a label DNA element to disrupt the function of a geneendogenous to the host organism, as the sole function of this DNA wouldbe to identify the origin of the organism. For example, one couldintroduce a unique DNA sequence into a plant and this DNA element wouldidentify all cells, plants, and progeny of these cells as having arisenfrom that labeled source. It is proposed that inclusion of label DNAswould enable one to distinguish proprietary germplasm or germplasmderived from such, from unlabelled germplasm.

Another possible element which may be introduced is a matrix attachmentregion element (MAR), such as the chicken lysozyme A element (Stief,1989), which can be positioned around an expressible gene of interest toeffect an increase in overall expression of the gene and diminishposition dependent effects upon incorporation into the plant genome(Stief et al., 1989; Phi-Van et al., 1990).

5. Other

Other examples of non-protein expressing sequences specificallyenvisioned for use with the invention include tRNA sequences, forexample, to alter codon usage, and rRNA variants, for example, which mayconfer resistance to various agents such as antibiotics.

XI. Biological Functional Equivalents

Modification and changes may be made in the centromeric DNA segments ofthe current invention and still obtain a functional molecule withdesirable characteristics. The following is a discussion based uponchanging the nucleic acids of a centromere to create an equivalent, oreven an improved, second-generation molecule.

In particular embodiments of the invention, mutated centromericsequences are contemplated to be useful for increasing the utility ofthe centromere. It is specifically contemplated that the function of thecentromeres of the current invention may be based upon the secondarystructure of the DNA sequences of the centromere, modification of theDNA with methyl groups or other adducts, and/or the proteins whichinteract with the centromere. By changing the DNA sequence of thecentromere, one may alter the affinity of one or morecentromere-associated protein(s) for the centromere and/or the secondarystructure or modification of the centromeric sequences, thereby changingthe activity of the centromere. Alternatively, changes may be made inthe centromeres of the invention which do not affect the activity of thecentromere. Changes in the centromeric sequences which reduce the sizeof the DNA segment needed to confer centromere activity are contemplatedto be particularly useful in the current invention, as would changeswhich increased the fidelity with which the centromere was transmittedduring mitosis and meiosis.

XII. Plants

The term “plant,” as used herein, refers to any type of plant. Theinventors have provided below an exemplary description of some plantsthat may be used with the invention. However, the list is not in any waylimiting, as other types of plants will be known to those of skill inthe art and could be used with the invention.

A common class of plants exploited in agriculture are vegetable crops,including artichokes, kohlrabi, arugula, leeks, asparagus, lettuce(e.g., head, leaf, romaine), bok choy, malanga, broccoli, melons (e.g.,muskmelon, watermelon, crenshaw, honeydew, cantaloupe), brusselssprouts, cabbage, cardoni, carrots, napa, cauliflower, okra, onions,celery, parsley, chick peas, parsnips, chicory, chinese cabbage,peppers, collards, potatoes, cucumber plants (marrows, cucumbers),pumpkins, cucurbits, radishes, dry bulb onions, rutabaga, eggplant,salsify, escarole, shallots, endive, garlic, spinach, green onions,squash, greens, beet (sugar beet and fodder beet), sweet potatoes, swisschard, horseradish, tomatoes, kale, turnips, and spices.

Other types of plants frequently finding commercial use include fruitand vine crops such as apples, apricots, cherries, nectarines, peaches,pears, plums, prunes, quince almonds, chestnuts, filberts, pecans,pistachios, walnuts, citrus, blueberries, boysenberries, cranberries,currants, loganberries, raspberries, strawberries, blackberries, grapes,avocados, bananas, kiwi, persimmons, pomegranate, pineapple, tropicalfruits, pomes, melon, mango, papaya, and lychee.

Many of the most widely grown plants are field crop plants such asevening primrose, meadow foam, corn (field, sweet, popcorn), hops,jojoba, peanuts, rice, safflower, small grains (barley, oats, rye,wheat, etc.), sorghum, tobacco, kapok, leguminous plants (beans,lentils, peas, soybeans), oil plants (rape, mustard, poppy, olives,sunflowers, coconut, castor oil plants, cocoa beans, groundnuts), fibreplants (cotton, flax, hemp, jute), lauraceae (cinnamon, camphor), orplants such as coffee, sugarcane, tea, and natural rubber plants.

Still other examples of plants include bedding plants such as flowers,cactus, succulents and ornamental plants, as well as trees such asforest (broad-leaved trees and evergreens, such as conifers), fruit,ornamental, and nut-bearing trees, as well as shrubs and other nurserystock.

XIII. Definitions

As used herein, the terms “autonomous replicating sequence” or “ARS” or“origin of replication” refer to an origin of DNA replication recognizedby proteins that initiate DNA replication.

As used herein, the terms “binary BAC” or “binary bacterial artificialchromosome” refer to a bacterial vector that contains the T-DNA bordersequences necessary for Agrobacterium mediated transformation (see, forexample, Hamilton et al., 1996; Hamilton, 1997; and Liu et al., 1999.

As used herein, the term “candidate centromere sequence” refers to anucleic acid sequence which one wishes to assay for potential centromerefunction.

As used herein, a “centromere” is any DNA sequence that confers anability to segregate to daughter cells through cell division. In onecontext, this sequence may produce a segregation efficiency to daughtercells ranging from about 1% to about 100%, including to about 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 95% of daughter cells.Variations in such a segregation efficiency may find importantapplications within the scope of the invention; for example, minichromosomes carrying centromeres that confer 100% stability could bemaintained in all daughter cells without selection, while those thatconfer 1% stability could be temporarily introduced into a transgenicorganism, but be eliminated when desired. In particular embodiments ofthe invention, the centromere may confer stable segregation of a nucleicacid sequence, including a recombinant construct comprising thecentromere, through mitotic or meiotic divisions, including through bothmeiotic and meitotic divisions. A plant centromere is not necessarilyderived from plants, but has the ability to promote DNA segregation inplant cells.

As used herein, the term “centromere-associated protein” refers to aprotein encoded by a sequence of the centromere or a protein which isencoded by host DNA and binds with relatively high affinity to thecentromere.

As used herein, the term “circular permutations” refer to variants of asequence that begin at base n within the sequence, proceed to the end ofthe sequence, resume with base number one of the sequence, and proceedto base n−1. For this analysis, n may be any number less than or equalto the length of the sequence. For example, circular permutations of thesequence ABCD are: ABCD, BCDA, CDAB, and DABC.

As used herein, the term “crop” includes any plant or portion of a plantgrown or harvested for commercial or beneficial purposes.

As used herein, “eukaryote” refers to living organisms whose cellscontain nuclei. A eukaryote may be distinguished from a “prokaryote”which is an organism which lacks nuclei. Prokaryotes and eukaryotesdiffer fundamentally in the way their genetic information is organized,as well as their patterns of RNA and protein synthesis.

As used herein, the term “expression” refers to the process by which astructural gene produces an RNA molecule, typically termed messenger RNA(mRNA). The mRNA is typically, but not always, translated intopolypeptide(s).

As used herein, the term “genome” refers to all of the genes and DNAsequences that comprise the genetic information within a given cell ofan organism. Usually, this is taken to mean the information containedwithin the nucleus, but also includes the organelles.

As used herein, the term “higher eukaryote” means a multicellulareukaryote, typically characterized by its greater complex physiologicalmechanisms and relatively large size. Generally, complex organisms suchas plants and animals are included in this category. Preferred highereukaryotes to be transformed by the present invention include, forexample, monocot and dicot angiosperm species, gymnosperm species, fernspecies, plant tissue culture cells of these species, animal cells andalgal cells. It will of course be understood that prokaryotes andeukaryotes alike may be transformed by the methods of this invention.

As used herein, the term “host” refers to any organism that contains aplasmid, expression vector, or integrated construct comprising a plantcentromere. Preferred examples of host cells for cloning, useful in thepresent invention, are bacteria such as Escherichia coli, Bacillussubtilis, Pseudomonas, Streptomyces, Salmonella, and yeast cells such asS. cerevisiae. Host cells which can be targeted for expression of a minichromosome may be plant cells of any source and specifically includeArabidopsis, maize, rice, sugarcane, sorghum, barley, soybeans, tobacco,wheat, tomato, potato, citrus, or any other agronomically orscientifically important species.

As used herein, the term “hybridization” refers to the pairing ofcomplementary RNA and DNA strands to produce an RNA-DNA hybrid, oralternatively, the pairing of two DNA single strands from geneticallydifferent or the same sources to produce a double stranded DNA molecule.

As used herein, the term “linker” refers to a DNA molecule, generally upto 50 or 60 nucleotides long and synthesized chemically, or cloned fromother vectors. In a preferred embodiment, this fragment contains one, orpreferably more than one, restriction enzyme site for a blunt-cuttingenzyme and a staggered-cutting enzyme, such as BamHI. One end of thelinker fragment is adapted to be ligatable to one end of the linearmolecule and the other end is adapted to be ligatable to the other endof the linear molecule.

As used herein, a “library” is a pool of random DNA fragments which arecloned. In principle, any gene can be isolated by screening the librarywith a specific hybridization probe (see, for example, Young et al.,1977). Each library may contain the DNA of a given organism inserted asdiscrete restriction enzyme-generated fragments or as randomly sheeredfragments into many thousands of plasmid vectors. For purposes of thepresent invention, E. coli, yeast, and Salmonella plasmids areparticularly useful when the genome inserts come from other organisms.

As used herein, the term “lower eukaryote” refers to a eukaryotecharacterized by a comparatively simple physiology and composition, andmost often unicellularity. Examples of lower eukaryotes includeflagellates, ciliates, and yeast.

As used herein, a “mini chromosome” is a recombinant DNA constructincluding a centromere and capable of transmission to daughter cells.Mini chromosome may remain separate from the host genome (as episomes)or may integrate into host chromosomes. The stability of this constructthrough cell division could range between from about 1% to about 100%,including about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% andabout 95%. The mini chromosome construct may be a circular or linearmolecule. It may include elements such as one or more telomeres, ARSsequences, and genes. The number of such sequences included is onlylimited by the physical size limitations of the construct itself. Itcould contain DNA derived from a natural centromere, although it may bepreferable to limit the amount of DNA to the minimal amount required toobtain a segregation efficiency in the range of 1-100%. The minichromosome could also contain a synthetic centromere composed of tandemarrays of repeats of any sequence, either derived from a naturalcentromere, or of synthetic DNA. The mini chromosome could also containDNA derived from multiple natural centromeres. The mini chromosome maybe inherited through mitosis or meiosis, or through both meiosis andmitosis. As used herein, the term mini chromosome specificallyencompasses and includes the terms “plant artificial chromosome” or“PLAC,” or engineered chromosomes or microchromosomes and all teachingsrelevant to a PLAC or plant artificial chromosome specifically apply toconstructs within the meaning of the term mini chromosome.

As used herein, by “mini chromosome-encoded protein” it is meant apolypeptide which is encoded by a sequence of a mini chromosome of thecurrent invention. This includes sequences such as selectable markers,telomeres, etc., as well as those proteins encoded by any other selectedfunctional genes on the mini chromosome.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant calli, and the like, as well as whole plantsregenerated therefrom.

As used herein, the term “plasmid” or “cloning vector” refers to aclosed covalently circular extrachromosomal DNA or linear DNA which isable to replicate in a host cell and which is normally nonessential tothe survival of the cell. A wide variety of plasmids and other vectorsare known and commonly used in the art (see, for example, Cohen et al.,U.S. Pat. No. 4,468,464, which discloses examples of DNA plasmids, andwhich is specifically incorporated herein by reference).

As used herein, a “probe” is any biochemical reagent (usually tagged insome way for ease of identification), used to identify or isolate agene, a gene product, a DNA segment or a protein.

As used herein, the term “recombination” refers to any genetic exchangethat involves breaking and rejoining of DNA strands.

As used herein the term “regulatory sequence” refers to any DNA sequencethat influences the efficiency of transcription or translation of anygene. The term includes, but is not limited to, sequences comprisingpromoters, enhancers and terminators.

As used herein, a “selectable marker” is a gene whose presence resultsin a clear phenotype, and most often a growth advantage for cells thatcontain the marker. This growth advantage may be present under standardconditions, altered conditions such as elevated temperature, or in thepresence of certain chemicals such as herbicides or antibiotics. Use ofselectable markers is described, for example, in Broach et al. (1979).Examples of selectable markers include the thymidine kinase gene, thecellular adenine-phosphoribosyltransferase gene and the dihydrylfolatereductase gene, hygromycin phosphotransferase genes, the bar gene andneomycin phosphotransferase genes, among others. Preferred selectablemarkers in the present invention include genes whose expression conferantibiotic or herbicide resistance to the host cell, sufficient toenable the maintenance of a vector within the host cell, and whichfacilitate the manipulation of the plasmid into new host cells. Ofparticular interest in the present invention are proteins conferringcellular resistance to ampicillin, chloramphenicol, tetracycline, G-418,bialaphos, and glyphosate for example.

As used herein, a “screenable marker” is a gene whose presence resultsin an identifiable phenotype. This phenotype may be observable understandard conditions, altered conditions such as elevated temperature, orin the presence of certain chemicals used to detect the phenotype.

As used herein, the term “site-specific recombination” refers to anygenetic exchange that involves breaking and rejoining of DNA strands ata specific DNA sequence.

As used herein, a “structural gene” is a sequence which codes for apolypeptide or RNA and includes 5′ and 3′ ends. The structural gene maybe from the host into which the structural gene is transformed or fromanother species. A structural gene will preferably, but not necessarily,include one or more regulatory sequences which modulate the expressionof the structural gene, such as a promoter, terminator or enhancer. Astructural gene will preferably, but not necessarily, confer some usefulphenotype upon an organism comprising the structural gene, for example,herbicide resistance. In one embodiment of the invention, a structuralgene may encode an RNA sequence which is not translated into a protein,for example a tRNA or rRNA gene.

As used herein, the term “telomere” refers to a sequence capable ofcapping the ends of a chromosome, thereby preventing degradation of thechromosome end, ensuring replication and preventing fusion to otherchromosome sequences. Telomeres can include naturally occurring telomeresequences or synthetic sequences. Telomres from one species may confertelomere activity in another species.

As used herein, the terms “transformation” or “transfection” refer tothe acquisition in cells of new DNA sequences through the chromosomal orextra-chromosomal addition of DNA. This is the process by which nakedDNA, DNA coated with protein, or whole mini chromosomes are introducedinto a cell, resulting in a potentially heritable change.

As used herein the term “consensus” refers to a nucleic acid sequencederived by comparing two or more related sequences. A consensus sequencedefines both the conserved and variable sites between the sequencesbeing compared. Any one of the sequences used to derive the consensus orany permutation defined by the consensus may be useful in constructionmini chromosomes.

As used herein the term “repeated nucleotide sequence” refers to anynucleic acid sequence of at least 25 bp present in a genome or arecombinant molecule that occurs at least two or more times and that arepreferably at least 80% identical either in head to tail or head to headorientation either with or without intervening sequence between repeatunits.

XIV. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skilled the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

Example 1 Isolation of Genomic DNA

Tissue from various plants are harvested for DNA extraction. For DNAextraction, leaf tissue is cooled in liquid nitrogen, ground to a finepowder and transferred to an organic solvent-resistant test tube orbeaker. Warm CTAB extraction solution (2% (w/v) CTAB, 100 mM Tris-Cl, pH9.5, 20 mM EDTA, pH 8.0, 1.4 M NaCl, 1% polyethylene gycol) is added ina ratio of 20 ml per gram of tissue and mixed thoroughly. For each 20 mlextraction buffer, 50 microliters of β-mercaptoethanol and 30microliters of 30 mg/ml RNAse A are added and the mixture is incubatedfor 10-60 min. at 65° C. with occasional mixing. The homogenate isextracted with an equal volume of chloroform, and is then centrifuged 5min at 7500×g (8000 rpm in JA20; 10,000 rpm in a microcentrifuge, forsmaller samples), 4° C. The top (aqueous) phase is recovered and nucleicacids are precipitated by adding 1 volume isopropanol. After mixing, theprecipitate is pelleted at 15 min at 7500×g, 4° C. The pellet is washedwith 70% ethanol, dried and resuspended in a minimal volume of TE (10 mMTris-Cl, pH 8.0, 0.1 mM EDTA, pH 8.0).

Example 2 Brassica oleracea Centromere Repeat Sequences

We purified repetitive sequences from Brassica oleracea (Brassicaoleracea fast plants, obtained from the Wisconsin Crucifer Cooperative).We set forth herein two centromere repeats, termed ChrBo1 and ChrBo2. Wedetermined the consensus of each repeat as described in Example 6.

The consensus sequence of ChrBo1 is shown in FIG. 1A (SEQ ID NO:1). Thisconsensus was assembled from DNA sequences collected by the inventors.Twenty-four of these sequences completely spanned the repeat, and nineothers partially covered the repeat. The length of this repeat is180±0.86 base pairs, and A and T comprise of 60% of the consensus.

The consensus sequence of ChrBo2 is shown in FIG. 1B (SEQ ID NO:2). Thisconsensus was assembled from DNA sequences collected by the inventors.Five of these sequences completely spanned the repeat, and two otherspartially covered the repeat. The length of this repeat is 180±0.45 basepairs, and A and T comprise 63% of the consensus.

The two repeats (ChrBo1 and ChrBo2) were aligned to each other using theClustalX program (ClustalX is a free multiple sequence alignment programfor Windows. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin,F. and Higgins, D. G. (1997) The ClustalX windows interface: flexiblestrategies for multiple sequence alignment aided by quality analysistools. Nucleic Acids Research, 24:4876-4882.). The two consensussequences differ significantly from each other at several bases. Thosesites with significant differences (chi-squared, P<0.05) are highlightedas shown in FIG. 1C.

The GenBank nt database and the plant satellite DNA database (PlantSat)were compared to the inventors' consensus sequences using the blastnprogram and an Expect value threshold score of −3. Consensus sequenceswere assembled using all inventors' and GenBank sequences that matchedwith an Expect (E) value of less than −45.

The revised consensus sequence of ChrBo1 is shown in FIG. 1D (SEQ IDNO:3). This consensus was assembled from thirty-three DNA sequencescollected by the inventors and eighteen GenBank sequences (Table 10).Thirty of these sequences completely spanned the repeat, and twenty-oneothers partially covered the repeat. The length of this repeat is180±0.81 base pairs, and A and T comprise of 59% of the consensus.

TABLE 1 GenBank sequences (accession numbers) that match inventors'ChrBo1 consensus M30962 M30963 M31436 M31435 M31438 M31434 M31439 M31437X68786 X12736 X07519 X16589 X15291 X68783 X68784 X61583 AJ228348 Z22947

The revised consensus sequence of ChrBo2 is shown in FIG. 1E (SEQ IDNO:4). This consensus was assembled from seven DNA sequences collectedby the inventors and five GenBank sequences (Table 2). Seven of thesesequences completely spanned the repeat, and five others partiallycovered the repeat. The length of this repeat is 180±0.44 base pairs,and A and T comprise of 63% of the consensus.

TABLE 2 GenBank sequences (accession numbers) that match inventors'ChrBo2 consensus AJ228347 M30962 X12736 X61583 X68785

The two revised consensus sequences (ChrBo1 and ChrBo2) were aligned toeach other using the ClustalX program. The two consensus sequencesdiffer significantly (chi-squared, P<0.05) from each other at severalbases (highlighted as shown in FIG. 1F).

A total of 20 GenBank entries match the Brassica oleracea centromeresequences defined by the inventors. These are annotated as follows:

Xle7-2EB gene

Xle4-7B gene

Xle6-14H gene

Satellite tandem repeat monomer

HindIII satellite repeat

Satellite DNA inverted direct repeat

Tandem repeated DNA

Highly repetitive DNA

They are not annotated as centromere repeats in GenBank. A completedlist of these sequences are shown in Table 3.

TABLE 3 GenBank entries match the Brassica oleracea centromere sequencesdefined by the inventors No. GenBank of Accession base % No. AnnotationRepeat Position pairs Identity X68786 B. juncea Xle7- Complete 472-651180 97 2EB gene X68786 B. juncea Xle7- Complete 763-942 180 94 2EB geneX68786 B. juncea Xle7- Partial 648-761 115 96 2EB gene X12736 B.campestries Complete 181-2  180 97 DNA for satellite tandem repeatmonomer (consensus sequence) X07519 Wild cabbage Complete 179-1  179 97satellite DNA X61583 B. napus Canrep Complete  2-173 176 98 highlyrepetitive DNA X68783 B. juncea repetitive Partial  2-173 172 97 DNAsequence canrep subfamily A X68784 B. juncea Xle4-7B Complete  983-1162180 95 gene X68784 B. juncea Xle4-7B Partial 815-986 172 94 geneAJ228348 B. carinata DNA, Partial  2-173 172 96 HindIII satellite repeat(clone pBcar3) M31438 B. oleracea satellite Partial 176-1  176 94 DNAinverted direct repeat X16589 B. nigra tandem Partial 177-1  177 94repeat DNA (clone BN1G 9, BN1G 23, BG1G 14) M31434 B. oleracea satellitePartial 176-8  169 95 DNA inverted direct repeat M31437 B. oleraceasatellite Partial 175-1  175 94 DNA inverted direct repeat M30963 B.juncea tandemly Complete 181-2  180 93 repeated DNA M31435 B. oleraceasatellite Partial 174-8  169 94 DNA inverted direct repeat M31436 B.oleracea satellite Partial 175-1  177 94 DNA inverted direct repeatX15291 B. juncea satellite Partial  1-161 161 95 DNA M31439 B. oleraceasatellite Partial 176-1  177 90 DNA inverted direct repeat Z22947 B.campestris Partial 181-347 170 90 satellite DNA Z22947 B. campestrisPartial  2-179 178 89 satellite DNA M30962 B. campestris Complete 181-2 180 87 tandemly repeated DNA X68785 B. juncea Xle6- Complete 580-758 18092 14H gene X68785 B. juncea Xle6- Partial 404-568 165 90 14H geneAJ228347 B. carinata DNA, Partial 177-2  176 90 HindIII satellite repeat(clone pBcar5)

Example 3 Glycine max Centromere Repeat Sequences

We purified repetitive sequences from soybean (Glycine max, varietyWilliams 82), and set forth herein two centromere repeats, termed ChrGm1and ChrGm2. We determined the consensus of each repeat as shown inExample 6.

The consensus sequence for ChrGm1 is shown in FIG. 2A (SEQ ID NO:5).This consensus was assembled from DNA sequences collected by theinventors. Seven of these sequences completely spanned the repeat, andtwenty-five others partially covered the repeat. It is 92±0.79 basepairs in length, and A and T comprise of 63% of the consensus.

The consensus sequence for ChrGm2 is shown in FIG. 2B (SEQ ID NO:6).This consensus was assembled from DNA sequences collected by theinventors. Ten of these sequences completely spanned the repeat, andeleven others partially covered the repeat. It is 91±0.48 base pairs inlength, and A and T comprise of 62% of the consensus.

The two repeats (ChrGm1 and ChrGm2) were aligned to each other using theClustalX program Those sites which differ significantly from each other(chi-squared, P<0.05) are highlighted in FIG. 2C.

The GenBank nt database and the plant satellite DNA database werecompared to the inventors' consensus sequences using the blastn programand an Expect value threshold of −3. Consensus sequences were builtusing all inventors' and GenBank sequences that matched with an Expect(E) value of less than −25.

The revised consensus sequence for ChrGm1 is shown in FIG. 2D (SEQ IDNO:7). This consensus was assembled from thirty-two DNA sequencescollected by the inventors and one matching sequence from GenBank(accession number Z26334). Eight of these sequences completely spannedthe repeat, and twenty-five others partially covered the repeat. It is92±0.74 base pairs in length, and A and T comprise of 56% of theconsensus.

The revised consensus sequence for ChrGm2 is shown in FIG. 2E (SEQ IDNO:8). This consensus was assembled from twenty-one DNA sequencescollected by the inventors and three matching sequences from GenBank(accession numbers AF297983, AF297984, AF297985). Ten of these sequencescompletely spanned the repeat, and fourteen others partially covered therepeat. It is 91±0.53 base pairs in length, and A and T comprise of 61%of the consensus.

The two repeats (ChrGm1 and ChrGm2) were aligned to each other using theClustalX program Those sites with significant differences (chi-squared,P<0.05) are highlighted in FIG. 2F.

A total of 4 GenBank entries match the Glycine max centromere sequencesdefined by the inventors. These are annotated as follows:

Satellite DNA

Tospovirus resistance protein C (Sw5-c), tospovirus resistance protein D(Sw5-d), and tospovirus resistance protein E (Sw5-e) genes

They are not annotated as centromere repeats in GenBank. A complete listof these sequences is shown in Table 4:

TABLE 4 GenBank entries match the Glycine max centromere sequencesdefined by the inventors GenBank No. of Accession base % No. AnnotationRepeat Position pairs Identity Z26334 G. max satellite Complete 92-1 9295 DNA AF297985 G. max clone Partial  259-173 87 93 TRS3 tandemrepetitive repeat region AF297985 G. max clone Partial 78-3 76 94 TRS3tandem repetitive repeat region AF297985 G. max clone Partial 168-83 8690 TRS3 tandem repetitive repeat region AF297984 G. max clone Partial170-84 87 91 TRS2 tandem repetitive repeat region AF297984 G. max clonePartial  260-175 86 88 TRS2 tandem repetitive repeat region AF297984 G.max clone Partial 79-3 77 89 TRS2 tandem repetitive repeat regionAF297983 G. max clone Partial 77-3 75 94 TRS1 tandem repetitive repeatregion

Example 4 Lycopersicon esculentum Centromere Repeat Sequences

We purified repetitive sequences from tomato (Lycopersicon esculentum,variety Microtom) and set forth herein one centromere repeat. Wedetermined the consensus of this repeat as shown in Example 6.

The consensus sequence of ChrLe1 is shown in FIG. 3A (SEQ ID NO:9). Thisconsensus was assembled from forty-two DNA sequences collected by theinventors. Eighteen of these sequences completely spanned the repeat,and twenty-four others partially covered the repeat. The repeat is181±0.61 base pairs in length, and A and T comprise of 50% of theconsensus.

The GenBank nt database and the plant satellite DNA database werecompared to the inventors' consensus sequences using the blastn programand an Expect value threshold value of −3. Consensus sequences werebuilt using all inventors' and GenBank sequences matched with an Expect(E) value of less than −40.

We determined the consensus of this repeat. The repeat is 181±0.61 basepairs in length, and A and T comprise of 50% of the consensus.

The revised consensus sequence of ChrLe1 is shown in FIG. 3B (SEQ IDNO:10). This consensus was assembled from forty-two sequences collectedby the inventors and two GenBank sequence. Eighteen of these sequencescompletely spanned the repeat, and twenty-six others partially coveredthe repeat. The GenBank sequences are accession numbers X87233 andAY007367.

Neither of the 2 GenBank entries that match the Lycopersicon esculentumcentromere sequences defined by the inventors are complete repeats; theymatch only a portion of the sequence identified by the company. Theseare annotated as follows:

Satellite DNA

Tandem Repetitive Repeat Region

They are not annotated as centromere repeats in GenBank. A complete listof these sequences is shown in Table 5.

TABLE 5 GenBank entries match the Lycopersicon esculentum centromeresequences defined by the inventors No. GenBank of Accession base % No.Annotation Repeat Position pairs Identity X87233 L. esculentum Partial163-1  161 93 satellite DNA AY007367 L. esculentum Partial 12003-12156154 93 tospovirus resistance protein C (Sw5-c), tospovirus resistanceprotein D (Sw5-d), and tospovirus resistance protein E (Sw5-e) genesAY007367 L. esculentum Partial 12184-12344 161 90 tospovirus resistanceprotein C (Sw5-c), tospovirus resistance protein D (Sw5-d), andtospovirus resistance protein E (Sw5-e) genes AY007367 L. esculentumPartial 12546-12700 155 90 tospovirus resistance protein C (Sw5-c),tospovirus resistance protein D (Sw5-d), and tospovirus resistanceprotein E (Sw5-e) genes AY007367 L. esculentum Partial 12365-12526 15789 tospovirus resistance protein C (Sw5-c), tospovirus resistanceprotein D (Sw5-d), and tospovirus resistance protein E (Sw5-e) genes

Example 5 Zea mays Centromere Repeat Sequences

We purified repetitive sequences from corn (Zea mays, variety B73), andset forth herein one centromere repeat, termed ChrZm1. We determined theconsensus of the repeat as shown in Example 5. The repeat is 180±1.15base pairs in length, and A and T comprise of 56% of the consensus.

The consensus sequence of ChrZm1 is shown in FIG. 4A (SEQ ID NO:11).This consensus was assembled from thirty-eight DNA sequences collectedby the inventors. Three of these sequences completely spanned therepeat, and thirty-five others partially covered the repeat.

The GenBank nt database and the plant satellite DNA database werecompared to the inventors' consensus sequences using the blastn programand an Expect value threshold score of −3. Consensus sequences werebuilt using all inventors' and GenBank sequences matched with an Expect(E) value of −50.

The revised consensus sequence of ChrZm1 is shown in FIG. 4B (SEQ IDNO:12). This consensus was assembled from thirty-eight DNA sequencescollected by the inventors and twenty-six matching GenBank sequences(Table 6). Twenty of these sequences completely spanned the repeat, andforty-four others partially covered the repeat. The length of the repeatis 180±0.51 base pairs, and A and T comprise the consensus.

TABLE 6 GenBank sequences that match the inventors' ChrZm1 consensusM32521 M32522 M32523 M32524 M32525 M32526 M32527 M32528 M32529 M32530M32531 M32532 M32533 M32534 M325375 M32536 M32537 M32538 M35408 AF030934AF030935 AF030936 AF030937 AF030938 AF030939 AF030940

A total of 26 GenBank entries match the Zea mays centromere sequencesdefined by the inventors. These are annotated as follows:

180-bp knob-specific repeat region

heterochromatin repetitive DNA

They are not annotated as centromere repeats in GenBank. A complete listof these sequences is shown in Table 7.

TABLE 7 GenBank entries match the Lycopersicon esculentum centromeresequences defined by the inventors No. GenBank of Accession base % No.Annotation Repeat Position pairs Identity M32522 Maize 180-bp Complete1-180 180 96 knob-specific repeat region M32521 Maize 180-bp Complete1-180 180 96 knob-specific repeat region M32533 Z. mays subsp. Complete1-180 180 96 mexicana 180-bp knob-specific repeat region M32525 Maize180-bp Complete 1-180 180 96 knob-specific repeat region M32524 Maize180-bp Complete 1-180 180 96 knob-specific repeat region M32523 Maize180-bp Complete 1-180 180 96 knob-specific repeat region M35408 CornComplete 1-180 180 96 heterochromatin repetitive DNA M32526 Maize 180-bpComplete 1-180 180 95 knob-specific repeat region AF030939 Z. mays180-bp Complete 1-180 180 95 knob-associated tandem repeat 15- T3-2M32528 Maize 180-bp Complete 1-180 180 95 knob-specific repeat regionM32534 Z. mays subsp. Complete 1-180 180 94 mexicana 180-bpknob-specific repeat region M32527 Maize 180-bp Partial 8-179 172 95knob-specific repeat region M32538 T. dactyloides Complete 1-179 179 94(Tripsacum dactyloides, gama grass) 180-bp knob-specific repeat regionM32529 Maize 180-bp Complete 1-180 180 93 knob-specific repeat regionAF030938 Z. mays 180-bp Partial 4-180 177 93 knob-associated tandemrepeat 15- T3-1 M32532 Maize 180-bp Complete 1-180 180 93 knob-specificrepeat region AF030937 Z. mays 180-bp Complete 1-180 180 92knob-associated tandem repeat 1- T7-2 AF030940 Z. mays 180-bp Complete1-180 180 92 knob-associated tandem repeat 15- T7-1 AF030936 Z. mays180-bp Partial 10-180  172 93 knob-associated tandem repeat 1- T7-1M32537 T. dactyloides 180- Complete 1-180 180 92 bp knob-specific repeatregion M32530 Maize 180-bp Complete 1-180 180 92 knob-specific repeatregion M32531 Maize 180-bp Complete 1-179, 180 91 knob-specificintroduced repeat region one gap AF030935 Z. mays 180-bp Partial 1-175175 90 knob-associated tandem repeat 1- T3-2 AF030934 Z. mays 180-bpPartial 47-201  155 92 knob-associated tandem repeat 1- T3-1 M32536 T.dactyloides 180- Complete 1-180 180 94 bp knob-specific repeat regionM32535 T. dactyloides 180- Complete 1-177, 2% 177 91 bp knob-specificgaps repeat region Six GenBank sequences of Zea mays centrometric repeatCentC were collected (Table 13) and assigned the identifier ChrZm2. Theconsensus of the repeat was determined as shown in Example 6. The repeatis 158 ± 1.6 base pairs in length. A and T comprises of 53% of thebases. All 6 sequences are of unit length.

The consensus sequence of ChrZm2 (SEQ ID NO:13) is shown in FIG. 4C.

TABLE 8 GenBank sequences of Zea mays centrometric repeat ChrZm2AF078918 AF078919 AF078920 AF0789121 AF078922 AF078923

Example 6 Determining Consensus Sequences

Sequences were first aligned and edited in Vector NTI suite7 (InforMax,7600 Wisconsin Ave., Suite 1100, Bethesda, Md. 20814) and exported as afasta file. A perl program, consensus.pl, was written and used todetermine the consensus for each position within the repeats based onthe following rules:

The most common base is designated as the consensus if it occurs threetimes more frequently than the second most common base.

If the occurrence of the most common base is not three times morefrequent than the second most common base, but the combined frequency ofthe two most common bases is three times that of the third most commonbase, and the frequency of the second most common base is greater thanthe frequency of the third most common base, then the second and firstbases are together considered as a consensus polymorphism, anddesignated using the IUPAC codes (M=A or C, R=A or G, W=A or T, S=C orG, Y=C or T, k=G or T, V=A or C or G, H=A or C or T, D=A or G or T, B=Cor G or T, N=G or T or C or A).

If the combined frequency of the two most common bases is not threetimes greater than that of the third most common base, but the combinedfrequency of the three most common bases is three times that of thefourth most common base, and the third most common base is more commonthan the fourth most common base, and the frequency of occurrence of thefourth base is less than or equal to 22%, the consensus is assignedaccording to the IUPAC ambiguity codes for the three most common bases.If the four bases occur approximately equally (23-27%), the consensus isassigned as N.

Example 7 Constructing BAC Vectors for Testing Centromere Function

A BAC clone may be retrofitted with one or more plant telomeres andselectable markers together with the DNA elements necessary forAgrobacterium transformation (FIG. 9). This method will provide a meansto deliver any BAC clone into plant cells and to test it for centromerefunction.

The method works in the following way. The conversion vector contains aretrofitting cassette. The retrofitting cassette is flanked by Tn10,Tn5, Tn7, Mu or other transposable elements and contains an origin ofreplication and a selectable marker for Agrobacterium, a plant telomerearray followed by T-DNA right and left borders followed by a secondplant telomere array and a plant selectable marker (FIG. 9). Theconversion vector is transformed into an E. coli strain carrying thetarget BAC. The transposable elements flanking the retrofitting cassettethen mediate transposition of the cassette randomly into the BAC clone.The retrofitted BAC clone can now be transformed into an appropriatestrain of Agrobacterium and then into plant cells where it can be testedfor high fidelity meiotic and mitotic transmission which would indicatethat the clone contained a complete functional plant centromere.

Example 8 Sequence Analysis of Arabidopsis Centromeres

A. Abundance of Genes in the Centromeric Regions

Expressed genes are located within 1 kb of essential centromeresequences in S. cerevisiae, and multiple copies of tRNA genes residewithin an 80 kb fragment necessary for centromere function in S. pombe(Kuhn et al., 1991). In contrast, genes are thought to be relativelyrare in the centromeres of higher eukaryotes, though there are notableexceptions. The Drosophila light, concertina, responder, and rolled lociall map to the centromeric region of chromosome 2, and translocationsthat remove light from its native heterochromatic context inhibit geneexpression. In contrast, many Drosophila and human genes that normallyreside in euchromatin become inactive when they are inserted near acentromere. Thus, genes that reside near centromeres likely have specialcontrol elements that allow expression (Karpen, 1994; Lohe and Hilliker,1995). The sequences of Arabidopsis CEN2 and CEN4, provided herein,provide a powerful resource for understanding how gene density andexpression correlate with centromere position and associated chromatin.

Annotation of chromosome II and IV(http://www.ncbi.nlm.nih.gov/Entrez/nucleotide.html) identified manygenes within and adjacent to CEN2 and CEN4 (FIG. 8, FIGS. 11A-11T). Thedensity of predicted genes on Arabidopsis chromosome arms averages 25per 100 kb, and in the repeat-rich regions flanking CEN2 and CEN4 thisdecreases to 9 and 7 genes per 100 kb, respectively (Bevan et al.,1999). Many predicted genes also reside within therecombination-deficient, genetically-defined centromeres. Within CEN2,there were 5 predicted genes per 100 kb; while CEN4 was strikinglydifferent, with 12 genes per 100 kb.

There was strong evidence that several of the predicted centromericgenes are transcribed. The phosphoenolpyruvate gene (CUE1) defines oneCEN5 border; mutations in this gene cause defects in light-regulatedgene expression (Li et al., 1995). Within the sequenced portions of CEN2and CEN4, 17% (27/160) of the predicted genes shared >95% identity withcloned cDNAs (ESTs), with three-fold more matches in CEN4 than in CEN2(http://www.tigr.org/tdb/at/agad/). Twenty-four of these genes havemultiple exons, and four correspond to single-copy genes with knownfunctions. A list of the predicted genes identified is given in Table 9,below. A list of additional genes encoded within the boundaries of CEN4are listed in Table 10. The identification of these genes is significantin that the genes may themselves contain unique regulatory elements ormay reside in genomic locations flanking unique control or regulatoryelements involved in centromere function or gene-expression. Inparticular, the current inventors contemplate use of these genes, or DNAsequences 0 to 5 kb upstream or downstream of these sequences, forinsertion into a gene of choice in a mini chromosome. It is expectedthat such elements could potentially yield beneficial regulatorycontrols of the expression of these genes, even when in the uniqueenvironment of a centromere.

To investigate whether the remaining 23 genes were uniquely encoded atthe centromere, a search was made in the database of annotated genomicArabidopsis sequences. With the exception of two genes, no homologswith >95% identity were found elsewhere in the 80% of the genome thathas been sequenced. The number of independent cDNA clones thatcorrespond to a single-copy gene provides an estimate of the level ofgene expression. On chromosome II, predicted genes with high qualitymatches to the cDNA database (>95% identity) match an average of fourindependent cDNA clones (range 1-78). Within CEN2 and CEN4, 11/27 genesexceed this average (Table 9). Finally, genes encoded at CEN2 and CEN4are not members of a single gene family, nor do they correspond to genespredicted to play a role in centromere functions, but instead havediverse roles.

Many genes in the Arabidopsis centromeric regions are nonfunctional dueto early stop codons or disrupted open reading frames, but fewpseudogenes were found on the chromosome arms. Though a large fractionof these pseudogenes have homology to mobile elements, many correspondto genes that are typically not mobile (FIGS. 11I-J and FIGS. 11S-T).Within the genetically-defined centromeres there were 1.0 (CEN2) and 0.7(CEN4) of these nonmobile pseudogenes per 100 kb; the repeat-richregions bordering the centromeres have 1.5 and 0.9 per 100 kbrespectively. The distributions of pseudogenes and transposable elementsare overlapping, indicting that DNA insertions in these regionscontributed to gene disruptions.

TABLE 9 Predicted genes within CEN2 and CEN4 that correspond to the cDNAdatabase. GenBank protein # of EST Putative function accession matches*CEN2 Unknown AAC69124 1 SH3 domain protein AAD15528 5 Unknown AAD15529 1unknown† AAD37022 1 RNA helicase‡ AAC26676 2 40S ribosomal protein S16AAD22696 9 CEN4 Unknown AAD36948 1 Unknown AAD36947 4 leucyl tRNAsynthetase AAD36946 4 aspartic protease AAD29758 6 Peroxisomal membraneprotein (PPM2)§ AAD29759 5 5′-adenylylsulfate reductase§ AAD29775 14symbiosis-related protein AAD29776 3 ATP synthase gamma chain 1 (APC1)§AAD48955 3 protein kinase and EF hand AAD03453 3 ABC transporterAAD03441 1 Transcriptional regulator AAD03444 14 Unknown AAD03446 12human PCF11p homolog AAD03447 6 NSF protein AAD17345 2 1,3-beta-glucansynthase AAD48971 2 pyridine nucleotide-disulphide AAD48975 4oxidoreductase Polyubiquitin (UBQ11)§ AAD48980 72 wound induced proteinAAD48981 6 short chain dehydrogenase/reductase AAD48959 7 SL15† AAD489392 WD40-repeat protein AAD48948 2 *Independent cDNAs with >95% identity,†related gene present in non-centromeric DNA, ‡potentially associatedwith a mobile DNA element, §characterized gene (B. Tugal, 1999; J. F.Gutierrez-Marcos, 1996; N. Inohara, 1991; J. Callis, 1995).

TABLE 10 List of additional genes encoded within the boundaries of CEN4.GenBank Nucleotide Putative Function accession Position3′(2′),5′-Bisphosphate Nucleotidase AC012392 71298-73681 Transcriptionalregulator AC012392 80611-81844 Equilibrative nucleoside transporter 1AC012392 88570-90739 Equilibrative nucleoside transporter 1 AC01239294940-96878 Equilibrative nucleoside transporter 1 AC012392 98929-101019 Equilibrative nucleoside transporter 1 AC012392113069-115262 unknown AC012392 122486-124729 4-coumarate--CoA ligaseAC012392 126505-128601 ethylene responsive protein AC012392130044-131421 Oxygen-evolving enhancer protein precursor AC012392134147-135224 Kinesin AC012392 137630-141536 receptor-like proteinkinase AC012392 141847-144363 LpxD-like protein AC012392 144921-146953hypersensitivity induced protein AC012392 147158-147838 ubiquitinAC012392 149057-149677 unknown AC012392 150254-151072 ubiquitin-likeprotein AC012392 153514-154470 ubiquitin-like protein AC012392155734-156513 ubiquitin-like protein AC012392 156993-157382 unknownAC012392 159635-165559 unknown AC012392 166279-166920 unknown AC012392167724-170212 ubiquitin-like protein AC012392 176819-178066polyubiquitin (UBQ10)§ AC012392 180613-182007phosphatidylinositol-3,4,5-triphosphate AC012477 89384-91291 bindingprotein Mitochondrial ATPase AC012477 94302-94677 RING-H2 finger proteinAC012477 95522-96142 unknown AC012477 104747-105196 Mitochondrial ATPaseAC012477 105758-106595 ferredoxin--NADP+ reductase AC012477107451-109095 unknown AC012477 109868-110620 U3 snoRNP-associatedprotein AC012477 111841-114133 UV-damaged DNA binding factor AC012477114900-121275 Glucan endo-1,3-Beta-Glucosidase precursor AC012477122194-122895 D123-like protein AC012477 125886-126887 AdrenodoxinPrecursor AC012477 127660-129246 N7 like-protein AC012477 129718-131012N7 like-protein AC012477 131868-133963 N7 like-protein AC012477134215-136569 N7 like-protein AC012477 139656-140864 §characterized gene(J. Callis, 1995).

B. Conservation of Centromeric DNA

To investigate the conservation of CEN2 and CEN4 sequences, PCR primerpairs were designed that correspond to unique regions in the Columbiasequence and used to survey the centromeric regions of Landsberg andColumbia at ˜20 kb intervals (FIGS. 13A, B). The primers used for theanalysis are listed in FIGS. 14A, B. Amplification products of theappropriate length were obtained in both ecotypes for most primer pairs.(85%), indicating that the amplified regions were highly similar. In theremaining cases, primer pairs amplified Columbia, but not Landsberg DNA,even at very low stringencies. In these regions, additional primers weredesigned to determine the extent of nonhomology. In addition to a largeinsertion of mitochondrial DNA in CEN2, two other non-conserved regionswere identified (FIGS. 13A, B). Because this DNA is absent fromLandsberg centromeres, it is unlikely to be required for centromerefunction; consequently, the relevant portion of the centromeric sequenceis reduced to 577 kb (CEN2) and 1250 kb (CEN4). The high degree ofsequence conservation between Landsberg and Columbia centromeresindicated that the inhibition of recombination frequencies was not dueto large regions of nonhomology, but instead was a property of thecentromeres themselves.

C. Sequence Similarity Between CEN2 and CEN4

In order to discern centromere function, a search was conducted fornovel sequence motifs shared between CEN2 and CEN4, excluding from thecomparison retroelements, transposons, characterized centromericrepeats, and coding sequences resembling mobile genes. After maskingsimple repetitive sequences, including homopolymer tracts andmicrosatellites, contigs of unique sequence measuring 417 kb and 851 kbfor CEN2 and CEN4, respectively, were compared with BLAST(http://blast.wustl.edu).

The comparison showed that the complex DNA within the centromere regionswas not homologous over the entire sequence length. However, 16 DNAsegments in CEN2 matched 11 regions in CEN4 with >60% identity. Thesequences were grouped into families of related sequences, and weredesignated AtCCS1-7 (Arabidopsis thaliana centromere conserved sequences1-7). These sequences were not previously known to be 20 repeated in theArabidopsis genome. The sequences comprised a total of 17 kb (4%) ofCEN2 DNA, had an average length of 1017 bp, and had an A+T content of65%. Based on similarity, the matching sequences were sorted intogroups, including two families containing 8 sequences each, 3 sequencesfrom a small family encoding a putative open reading frame, and 4sequences found once within the centromeres, one of which 25 correspondsto predicted CEN2 and CEN4 proteins with similarity throughout theirexons and introns.

Searches of the Arabidopsis genomic sequence database demonstrated thatAtCCS1-AtCCS5 were moderately repeated sequences that appear incentromeric and pericentromeric regions. The remaining sequences werepresent only in the genetically-defined centromeres. Similar comparisonsof all 16 S. cerevisiae centromeres defined a consensus consisting of aconserved 8 bp CDEI motif, an AT-rich 85 bp CDEII element, and a 26 bpCDEII region with 7 highly conserved nucleotides (Fleig et al., 1995).In contrast, surveys of the three S. pombe centromeres revealedconservation of overall centromere structure, but no universallyconserved motifs (Clark, 1998).

Example 9 Construction of Plant Mini Chromosomes

Mini chromosomes are constructed by combining the previously isolatedessential chromosomal elements. Exemplary mini chromosome vectorsinclude those designed to be “shuttle vectors”; i.e., they can bemaintained in a convenient host (such as E. coli, Agrobacterium oryeast) as well as plant cells.

A. General Techniques for Mini Chromosome Construction

A mini chromosome can be maintained in E. coli or other bacterial cellsas a circular molecule by placing a removable stuffer fragment betweenthe telomeric sequence blocks. The stuffer fragment is a dispensable DNAsequence, bordered by unique restriction sites, which can be removed byrestriction digestion of the circular DNAs to create linear moleculeswith telomeric ends. The linear mini chromosome can then be isolated by,for example, gel electrophoresis. In addition to the stuffer fragmentand the plant telomeres, the mini chromosome contains a replicationorigin and selectable marker that can function in plants to allow thecircular molecules to be maintained in bacterial cells. The minichromosomes also include a plant selectable marker, a plant centromere,and a plant ARS to allow replication and maintenance of the DNAmolecules in plant cells. Finally, the mini chromosome includes severalunique restriction sites where additional DNA sequence inserts can becloned. The most expeditious method of physically constructing such amini chromosome, i.e., ligating the various essential elements togetherfor example, will be apparent to those of ordinary skill in this art.

A number of mini chromosome vectors have been designed by the currentinventors and are disclosed herein for the purpose of illustration(FIGS. 7A-7H). These vectors are not limiting however, as it will beapparent to those of skill in the art that many changes and alterationsmay be made and still obtain a functional vector.

B. Modified Technique for Mini Chromosome Construction

A two step method was developed for construction of mini chromosomes,which allows adding essential elements to BAC clones containingcentromeric DNA. These procedures can take place in vivo, eliminatingproblems of chromosome breakage that often happen in the test tube. Thedetails and advantages of the techniques are as follows:

-   -   1.) One plasmid can be created that contains markers, origins        and border sequences for Agrobacterium transfer, markers for        selection and screening in plants, plant telomeres, and a loxP        site or other site useful for site-specific recombination in        vivo or in vitro. The second plasmid can be an existing BAC        clone, isolated from the available genomic libraries (FIG. 10A).    -   2.) The two plasmids are mixed, either within a single E. coli        cell, or in a test tube, and the site-specific recombinase cre        is introduced. This will cause the two plasmids to fuse at the        loxP sites (FIG. 10B).    -   3.) If deemed necessary, useful restriction sites (AseI/PacI or        Not I) are included to remove excess material. (for example        other selectable markers or replication origins)    -   4.) Variations include vectors with or without a Kan^(R) gene        (FIGS. 10B, 10C), with or without a LAT52 GUS gene, with a LAT52        GFP gene, and with a GUS gene under the control of other plant        promoters. (FIGS. 10C, 10D and 10E).

C. Method for Preparation of Stable Non-Integrated Mini chromosomes

A technique has been developed to ensure that mini chromosomes do notintegrate into the host genome (FIG. 10F). In particular, minichromosomes must be maintained as distinct elements separate from thehost chromosomes. In one method for ensuring that the introduced minichromosome does not integrate, the inventors envision a variety thatwould encode a lethal plant gene (such as diptheria toxin or any othergene product that, when expressed, causes lethality in plants). Thisgene could be located between the right Agrobacterium border and thetelomere. Mini chromosomes that enter a plant nucleus and integrate intoa host chromosome would result in lethality. However, if the minichromosome remains separate, and further, if the ends of this constructare degraded up to the telomeres, then the lethal gene would be removedand the cells would survive.

It should be understood that various changes and modifications to thepresently preferred embodiments described herein will be apparent tothose skilled in the art. Such changes and modifications can be madewithout departing from the spirit and scope of the present invention andwithout diminishing its intended advantages. It is therefore intendedthat such changes and modifications be covered by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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The invention claimed is:
 1. A method for generating revenue based on agricultural products comprising the steps of: developing a new crop comprising a mini-chromosome including a sequence of a crop plant centromere, wherein the crop plant centromere: (i) comprises n copies of a repeated nucleotide sequence from centromeric genomic DNA of the plant, wherein the repeated nucleotide sequence is about 75 base pairs to about 210 base pairs, and wherein n is at least 5; and (ii) confers an ability to a nucleic acid comprising the plant centromere to segregate to daughter cells through cell division; and receiving a fee for the new crop.
 2. The method of claim 1 wherein the fee is received as a royalty payment.
 3. The method of claim 1 wherein the minichromosome includes at least one gene that adds a desirable characteristic to the new crop.
 4. The method of claim 1 wherein the minichromosome includes at least two genes not present in an original crop containing the sequence.
 5. The method of claim 1 wherein the fee is received, at least in part, as a cross-license of rights.
 6. The method of claim 1 wherein the fee is received at least in part, as a service.
 7. A method for providing a service comprising: (a) identifying a sequence comprising a centromere of a commercial or crop plant, wherein the centromere (i) comprises n copies of a repeated nucleotide sequence from centromeric genomic DNA of the commercial or crop plant, wherein the repeated nucleotide sequence is about 75 base pairs to about 210 base pairs, and wherein n is at least 5; and (ii) confers an ability to a nucleic acid comprising the commercial or crop plant centromere to a nucleic acid comprising the commercial or crop plant centromere to segregate to daughter cells through cell division; (b) receiving a fee for the service, wherein the fee is one selected from the group consisting of (i) a royalty payment, (ii) a fee based on a sale of a product generated by a plant modified with the identified commercial plant or crop centromere, (iii) a fee received through sales of seed generated by a plant modified with the identified commercial plant or crop centromere; (iv) a fee based on a license for a new crop or product modified with the identified commercial or crop plant centromere, (v) a fee based on receipt of the identified commercial plant or crop centromere sequence, and (vi) a fee based on receipt of a product or service comprising identifying the commercial plant or crop centromere; (c) designing a plant minichromosome, comprising the centromere; and (d) constructing the plant minichromosome.
 8. The method of claim 7, wherein the service further comprises (e) contacting a starting plant cell with the plant minichromosome; and (f) transforming the starting plant cell with the plant minichromosome.
 9. The method of claim 8, further comprising (g) generating a plant from the transformed starting plant cell.
 10. The method of claim 9, wherein the service further comprises (h) providing a tissue or a seed from the generated plant.
 11. The method of claim 9, wherein the service further comprises (i) processing the tissue or seed from the generated plant to extract a product produced by the generated plant.
 12. The method of claim 1 or 7, wherein the plant centromere is from a monocotyledonous plant.
 13. The method of claim 12, wherein the monocotyledonous plant is selected from the group consisting of wheat, maize, rye, rice, turfgrass, oat, barley, sorghum, sugar cane, and millet.
 14. The method of claim 1 or 7, wherein the plant centromere is from a dicotyledonous plant.
 15. The method of claim 14, wherein the dicotyledonous plant is selected from the group consisting of tobacco, tomato, potato, pea, carrot, cauliflower, broccoli, soybean, canola, sunflower, alfalfa, and cotton.
 16. The method of claim 7, wherein the fee is a royalty payment, a one-time payment, or is a fee based on sales of at least one selected from the group consisting of seed comprising the minichromosome, a plant comprising the minichromosome, and a product made from the plant comprising the minichromosome.
 17. The method of any one of claims 7-9, wherein the plant minichromosome further comprises a nucleic acid sequence that encodes a commercially desirable trait. 